MN gene and protein

ABSTRACT

Identified herein is the location of the MN protein binding site, and MN proteins/polypeptides that compete for attachment to vertebrate cells with immobilized MN protein. Such MN proteins/polypeptides prevent cell-cell adhesion and the formation of intercellular contacts. The MN protein binding site is a therapeutic target that can be blocked by organic or inorganic molecules, preferably organic molecules, more preferably proteins/polypeptides that specifically bind to that site. Therapeutic methods for inhibiting the growth of preneoplastic/neoplastic vertebrate cells that abnormally express MN protein are disclosed. Vectors are provided that encode the variable domains of MN-specific antibodies and a flexible linker polypeptide separating those domains. Further vectors are disclosed that encode a cytotoxic protein/polypeptide operatively linked to the MN gene promoter or a MN gene promoter fragment comprising the HIF-1 consensus binding sequence, and which vectors preferably further encode a cytokine. The MN gene promoter is characterized, and the binding site for a repressor of MN transcription is disclosed. Further, the hypoxia inducibility of the MN gene and the uses of such inducibility are disclosed.

This application is a continuation of U.S. application Ser. No.11/356,568, filed Feb. 17, 2006 which is a continuation of now abandonedU.S. application Ser. No. 10/319,003, filed on Dec. 13, 2002. Thisapplication claims priority from U.S. Ser. No. 11/356,568 filed on Feb.17, 2006, U.S. Ser. No. 10/319,033 filed on Dec. 13, 2002 and from U.S.Provisional Application No. 60/341,036 filed on Dec. 13, 2001.

REFERENCE TO SEQUENCE LISTING

The Sequence Listing, filed electronically and identified asUSSN-11-932944-SEQ-LISTING, was created on Jan. 22, 2008, is 80.4 kb insize and is hereby incorporated by reference.

FIELD OF THE INVENTION

The present invention is in the general area of medical genetics and inthe fields of biochemical engineering, immunochemistry and oncology.More specifically, it relates to the MN gene—a cellular gene consideredto be an oncogene, known alternatively as MN/CA 9, CA 9, or carbonicanhydrase 9, which gene encodes the oncoprotein now known alternativelyas the MN protein, the MN/CA IX isoenzyme, MN/CA IX, carbonic anhydraseIX, or the MN/G250 protein.

BACKGROUND OF THE INVENTION

Zavada et al., International Publication Number WO 93/18152 (published16 Sep. 1993) and U.S. Pat. No. 5,387,676 (issued Feb. 7, 1995),describe the discovery and biological and molecular nature of the MNgene and protein. The MN gene was found to be present in the chromosomalDNA of all vertebrates tested, and its expression to be stronglycorrelated with tumorigenicity.

The MN protein was first identified in HeLa cells, derived from a humancarcinoma of cervix uteri. It is found in many types of human carcinomas(notably uterine cervical, ovarian, endometrial, renal, bladder, breast,colorectal, lung, esophageal, and prostate, among others). Very fewnormal tissues have been found to express MN protein to any significantdegree. Those MN-expressing normal tissues include the human gastricmucosa and gallbladder epithelium, and some other normal tissues of thealimentary tract. Paradoxically, MN gene expression has been found to belost or reduced in carcinomas and other preneoplastic/neoplasticdiseases in some tissues that normally express MN, e.g., gastric mucosa.

In general, oncogenesis may be signified by the abnormal expression ofMN protein. For example, oncogenesis may be signified: (1) when MNprotein is present in a tissue which normally does not express MNprotein to any significant degree; (2) when MN protein is absent from atissue that normally expresses it; (3) when MN gene expression is at asignificantly increased level, or at a significantly reduced level fromthat normally expressed in a tissue; or (4) when MN protein is expressedin an abnormal location within a cell.

Zavada et al., WO 93/18152 and Zavada et al., WO 95/34650 (published 21Dec. 1995) disclose how the discovery of the MN gene and protein and thestrong association of MN gene expression and tumorigenicity led to thecreation of methods that are both diagnostic/prognostic and therapeuticfor cancer and precancerous conditions. Methods and compositions wereprovided therein for identifying the onset and presence of neoplasticdisease by detecting or detecting and quantitating abnormal MN geneexpression in vertebrates. Abnormal MN gene expression can be detectedor detected and quantitated by a variety of conventional assays invertebrate samples, for example, by immunoassays using MN-specificantibodies to detect or detect and quantitate MN antigen, byhybridization assays or by PCR assays, such as RT-PCR, using MN nucleicacids, such as, MN cDNA, to detect or detect and quantitate MN nucleicacids, such as, MN mRNA.

Zavada et al, WO 93/18152 and WO 95/34650 describe the production ofMN-specific antibodies. A representative and preferred MN-specificantibody, the monoclonal antibody M75 (Mab M75), was deposited at theAmerican Type Culture Collection (ATCC) in Manassus, Va. (USA) underATCC Number HB 11128. The M75 antibody was used to discover and identifythe MN protein and can be used to identify readily MN antigen in Westernblots, in radioimmunoassays and immunohistochemically, for example, intissue samples that are fresh, frozen, or formalin-, alcohol-, acetone-or otherwise fixed and/or paraffin-embedded and deparaffinized. Anotherrepresentative and preferred MN-specific antibody, Mab MN12, is secretedby the hybridoma MN 12.2.2, which was deposited at the ATCC under thedesignation HB 11647. Example 1 of Zavada et al., WO 95/34650 providesrepresentative results from immunohistochemical staining of tissuesusing MAb M75, which results support the designation of the MN gene asan oncogene.

Many studies have confirmed the diagnostic/prognostic utility of MN. Thefollowing articles discuss the use of the MN-specific MAb M75 indiagnosing/prognosing precancerous and cancerous cervical lesions: Leff,D. N., “Half a Century of HeLa Cells: Transatlantic Antigen EnhancesReliability of Cervical Cancer Pap Test, Clinical Trials Pending,”BioWorld® Today: The Daily Biotechnology Newspaper, 9(55) (Mar. 24,1998); Stanbridge, E. J., “Cervical marker can help resolve ambigous Papsmears,” Diagnostics Intelligence, 10(5): 11 (1998); Liao andStanbridge, “Expression of the MN Antigen in Cervical PapanicolaouSmears Is an Early Diagnostic Biomarker of Cervical Dysplasia,” CancerEpidemiology, Biomarkers & Prevention, 5: 549-557 (1996); Brewer et al.,“A Study of Biomarkers in Cervical Carcinoma and Clinical Correlation ofthe Novel Biomarker MN,” Gynecologic Oncology, 63: 337-344 (1996); andLiao et al., “Identification of the MN Antigen as a Diagnostic Biomarkerof Cervical Intraepithelial Squamous and Glandular Neoplasia andCervical Carcinomas,” American Journal of Pathology, 145(3): 598-609(1994).

Premalignant and Malignant Colorectal Lesions. MN has been detected innormal gastric, intestinal, and biliary mucosa. [Pastorekova et al.,Gastroenterology, 112: 398-408 (1997).] Immunohistochemical analysis ofthe normal large intestine revealed moderate staining in the proximalcolon, with the reaction becoming weaker distally. The staining wasconfined to the basolateral surfaces of the cryptal epithelial cells,the area of greatest proliferative capacity. As MN is much more abundantin the proliferating cryptal epithelium than in the upper part of themucosa, it may play a role in control of the proliferation anddifferentiation of intestinal epithelial cells. Cell proliferationincreases abnormally in premalignant and malignant lesions of thecolorectal epithelium, and therefore, is considered an indicator ofcolorectal tumor progression. [Risio, M., J. Cell Biochem. 16G: 79-87(1992); and Moss et al., Gastroenterology, 111: 1425-1432 (1996).]

The MN protein is now considered to be the first tumor-associatedcarbonic anhydrase (CA) isoenzyme that has been described. Carbonicanhydrases (CAs) form a large family of genes encoding zincmetalloenzymes of great physiological importance. As catalysts ofreversible hydration of carbon dioxide, these enzymes participate in avariety of biological processes, including respiration, calcification,acid-base balance, bone resorption, formation of aqueous humor,cerebrospinal fluid, saliva and gastric acid [reviewed in Dodgson etal., The Carbonic Anhydrases, Plenum Press, New York-London, pp. 398(1991)]. CAs are widely distributed in different living organisms.

In mammals, at least seven isoenzymes (CA I-VII) and a few CA-relatedproteins (CARP/CA VIII, RPTP-β, RPTP-T) had been identified[Hewett-Emmett and Tashian, Mol. Phyl. Evol., 5: 50-77 (1996)], whenanalysis of the MN deduced amino acid sequence revealed a strikinghomology between the central part of the MN protein and carbonicanhydrases, with the conserved zinc-binding site as well as the enzyme'sactive center. Then MN protein was found to bind zinc and to have CAactivity. Based on that data, the MN protein is now considered to be theninth carbonic anhydrase isoenzyme—MN/CA IX. [Opavsky et al., Genomics,33: 480-487 (May 1996)]. [See also, Hewett-Emmett, supra, wherein CA IXis suggested as a nomenclatural designation.]

CAs and CA-related proteins show extensive diversity in both theirtissue distribution and in their putative or established biologicalfunctions [Tashian, R. E., Adv. in Genetics, 30: 321-356 (1992)]. Someof the CAs are expressed in almost all tissues (CA II), while theexpression of others appears to be more restricted (CA VI and CA VII insalivary glands). In cells, they may reside in the cytoplasm (CA I, CAII, CA III, and CA VII), in mitochondria (CA V), in secretory granules(CA VI), or they may associate with membrane (CA IV). Occasionally,nuclear localization of some isoenzymes has been noted [Parkkila et al.,Gut, 35: 646-650 (1994); Parkkilla et al., Histochem. J., 27: 133-138(1995); Mori et al., Gastroenterol., 105: 820-826 (1993)].

The CAs and CA-related proteins also differ in kinetic properties andsusceptibility to inhibitors [Sly and Hu, Annu. Rev. Biochem., 64:375-401 (1995)]. In the alimentary tract, carbonic anhydrase activity isinvolved in many important functions, such as saliva secretion,production of gastric acid, pancreatic juice and bile, intestinal waterand ion transport, fatty acid uptake and biogenesis in the liver. Atleast seven CA isoenzymes have been demonstrated in different regions ofthe alimentary tract. However, biochemical, histochemical andimmunocytochemical studies have revealed a considerable heterogeneity intheir levels and distribution [Swensen, E. R., “Distribution andfunctions of carbonic anhydrase in the gastrointestinal tract,” In: TheCarbonic Anhydrases, Cellular Physiology and Molecular Genetics,(Dodgson et al. eds.) Plenum Press, New York, pages 265-287 (1991); andParkkila and Parkkila, Scan J. Gastroenterol., 31: 305-317 (1996)].While CA II is found along the entire alimentary canal, CA IV is linkedto the lower gastrointestinal tract, CA I, III and V are present in onlya few tissues, and the expression of CA VI and VII is restricted tosalivary glands [Parkkila et al., Gut, 35: 646-650 (1994); Fleming etal., J. Clin. Invest., 96: 2907-2913 (1995); Parkkila et al.,Hepatology, 24: 104 (1996)].

MN/CA IX has a number of properties that distinguish it from other knownCA isoenzymes and evince its relevance to oncogenesis. Those propertiesinclude its density dependent expression in cell culture (e.g., HeLacells), its correlation with the tumorigenic phenotype of somatic cellhybrids between HeLa and normal human fibroblasts, its close associationwith several human carcinomas and its absence from corresponding normaltissues [e.g., Zavada et al., Int. J. Cancer, 54: 268-274 (1993);Pastorekova et al., Virology, 187: 620-626 (1992); Liao et al., Am. J.Pathol., 145: 598-609 (1994); Pastorek et al., Oncogene, 9: 2788-2888(1994); Cote, Women's Health Weekly: News Section, p. 7 (Mar. 30, 1998);Liao et al., Cancer Res., 57: 2827 (1997); Vermylen et al., “Expressionof the MN antigen as a biomarker of lung carcinoma and associatedprecancerous conditions,” Proceedings AACR, 39: 334 (1998); McKiernan etal., Cancer Res., 57: 2362 (1997); and Turner et al., Hum. Pathol.,28(6): 740 (1997)]. In addition, the in vitro transformation potentialof MN/CA IX cDNA has been demonstrated in NIH 3T3 fibroblasts [Pastoreket al., id.].

The MN protein has also been identified with the G250 antigen. Uemura etal., “Expression of Tumor-Associated Antigen MN/G250 in UrologicCarcinoma: Potential Therapeutic Target,” J. Urol., 157 (4 Suppl.): 377(Abstract 1475; 1997) states: “Sequence analysis and database searchingrevealed that G250 antigen is identical to MN, a human tumor-associatedantigen identified in cervical carcinoma (Pastorek et al., 1994).”

MN/CA IX has been identified as a novel hypoxia regulated marker ininvasive breast cancer as reported in Chia et al., “PrognosticSignificance of a Novel Hypoxia Regulated Marker, Carbonic Anhydrase IX(MN/CAIX) in Invasive Breast Cancer,” Breast Cancer Research andTreatment, 64(1): 43 (November 2000). Chia et al. stated “that MN/CA IXexpression is significantly increased in hypoxic conditions acrossvarious cell lines.” MN/CA IX expression was “found to be significantlyassociated with a higher tumor grade (p=0.003), a negative estrogenreceptor status (P<0.001) and tumor necrosis (p<0.001) . . . associatedwith significantly worst relapse-free survival (p=0.004) and a worseoverall survival (p=0.001).”

Hypoxia is a reduction in the normal level of tissue oxygen tension. Itoccurs during acute and chronic vascular disease, pulmonary disease andcancer, and produces cell death if prolonged. Pathways that areregulated by hypoxia include angiogenesis, glycolysis, growth-factorsignaling, immortalization, genetic instability, tissue invasion andmetastasis, apoptosis and pH regulation. [Harris, A. L., Nature Reviews,2: 38-47 (January 2002).]

Tumors become hypoxic because new blood vessels that develop in thetumors are aberrant and have poor blood flow. Although hypoxia is toxicto both tumor cells and normal cells, tumor cells undergo genetic andadaptive changes that allow them to survive and even proliferate in ahypoxic environment. These processes contribute to the malignantphenotype and to aggressive tumor behavior. Hypoxia is associated withresistance to radiation therapy and chemotherapy, but is also associatedwith poor outcome regardless of treatment modality, indicating that itmight be an important therapeutic target. Additionally, there is a needto find an alternative to the current Eppendorf pO₂ histograph methodfor assessing tumor hypoxia in patients. Although the Eppendorf methodprovides prognostic information in a variety of tumor types, it islimited to tumors acceptable for microneedle insertion. [Harris, A. L.,id.]

The central mediator of transcriptional up-regulation of a number ofgenes during hypoxia is the transcription factor HIF-1. HIF-1 is aheterodimer that consists of the hypoxic response factor HIF-1α and theconstitutively expressed aryl hydrocarbon receptor nuclear translocator(ARNT, also known as HIF-1β). In the absence of oxygen, HIF-1 binds toHIF-binding sites within hypoxia-response elements (HREs) ofoxygen-regulated genes, thereby activating the expression of numeroushypoxia-response genes, such as erythropoietin (EPO), and theproangiogenic growth factor vascular endothelial growth factor (VEGF).

Semenza et al. PNAS (USA), 88: 5680-5684 (1991) first identifiedcis-activating DNA sequences that function as tissue-specifichypoxia-inducible enhancers of human erythropoietin expression. Pugh etal., PNAS (USA), 88: 10533-71 (1991) isolated such a DNA sequence 3′ tothe mouse erythropoietin gene which acts as a hypoxia-inducible enhancerfor a variety of heterologous promoters. Maxwell et al., PNAS (USA), 90:2423-2427 (1993) have shown that the oxygen-sensing system whichcontrols erythropoietin expression is widespread in mammalian cells.

McBurney et al., Nucleic Acids Res., 19: 5755-61 (1991) found thatrepeating the hypoxia response element (HRE) sequence, located 5′ to thehypoxia-inducible mouse phosphoglycerate kinase gene (PGK), leads toincreased induction of the gene, and that using the interleukin-2 geneunder tissue-specific promoters can be used for specific targeting oftumors.

Hypoxia can be used to activate therapeutic gene delivery to specificareas of tissue. Dachs et al. “Targeting gene expression to hypoxictumor cells,” Nat. Med., 3: 515-20 (1997) has used the HRE from themouse PGK gene promoter to drive expression of heterologous genes bothin vitro and in vivo with controlled hypoxia.

For some HIF targets such as VEGF, a clear function in promoting tumorgrowth is established. [Kim et al., “Inhibition of vascular endothelialgrowth factor-induced angiogenesis suppresses tumour growth in vivo,”Nature (Lond.), 362: 841-844 (1993).] However, the full range of HIFtarget genes has not yet been defined, and identification of additionalgenes responding to this pathway is likely to provide further insightsinto the consequences of tumor hypoxia and HIF activation.

Indirect support for the importance of microenvironmental activation ofHIF has also been provided by recent demonstrations of constitutiveactivation of HIF after inactivation of the VHL tumor suppressor gene.[Maxwell et al., “The tumour suppressor protein VHL targetshypoxia-inducible factors for oxygen-dependent proteolysis,” Nature(Lond.), 399: 271-275 (1999)] and amplification of the HIF response byother oncogenic mutations. [Jiang et al., “V-SRC induces expression ofhypoxia-inducible factor 1 (HIF-1) and transcription of genes encodingvascular endothelial growth factor and enolase 1: involvement of HIF-1in tumor progression,” Cancer Res., 57: 5328-5335 (1997); Blagosklonnyet al., “p53 inhibits hypoxia-inducible factor-stimulatedtranscription,” J. Biol. Chem., 273: 11995-11998 (1998); Ravi et al.,“Regulation of tumor angiogenesis by p53-induced degradation ofhypoxia-inducible factor 1α,” Genes Dev., 14: 34-44 (2000); Zundel etal., “Loss of PTEN facilitates HIF-1 mediated gene expression,” GenesDev., 14: 391-396 (2000).]

Mutations in VHL cause the familial syndrome and are also found in themajority of sporadic RCCs. [Gnarra et al., “Mutations of the VHL tumoursuppressor gene in renal carcinoma,” Nat Genet., 7: 85-90 (1994).] Thegene product pVHL forms part of ubiquitin-ligase complex, [Lisztwan etal., “The von Hippel-Landau tumor suppressor protein is a component ofan E3 ubiquitin-protein ligase activity,” Genes Dev., 13: 1822-1833(1999); Iwai et al., “Identification of the von Hippel-Lindautumor-suppressor protein as part of an active E3 ubiquitin ligasecomplex,” Proc. Natl Acad. Sci (USA) 96: 12436-12441 (1999)] thattargets HIF-α subunits for oxygen-dependent proteolysis. [Maxwell etal., (1999) supra; Cockman et al., “Hypoxia inducible factor-α bindingand ubiquitination by the von Hippel-Landau tumor suppressor protein,”J. Biol. Chem., 275: 25733-25741 (2000).]

In VHL-defective cells, HIF-α is stabilized constitutively, resulting inup-regulation of hypoxia-inducible genes such as VEGF. [Maxwell et al.,(1999) supra.] Although the pVHL ubiquitinligase complex may have othertargets [Iwai et al., supra] and other functions of pVHL have beenproposed that may contribute to tumor suppressor effects [Pause et al.,“The von Hippel-Lindau tumor suppressor gene is required for cell cycleexit on serum withdrawal,” Proc. Natl. Acad. Sci. (USA) 95: 993-998(1998); Ohh et al., “The von Hippel-Landau tumor suppressor protein isrequired for proper assembly of an extracellular fibronectin matrix,”Mol. Cell, 1: 959-968 (1998)], these recent findings raise importantquestions as to the range of genes affected by constitutive HIFactivation and role of such genes in oncogenesis.

In that respect, MN/CA 9 considered to be an oncogene has an interestingposition as a transmembrane carbonic anhydrase (CA). CAs catalyze thereversible hydration of carbon dioxide to carbonic acid [Sly et al.,Annu. Rev. Biochem., 64: 375-401 (1995)], providing a potential linkbetween metabolism and pH regulation. One aspect of this invention isthe relationship between MN/CA 9 and hypoxia. MN/CA IX is shown to beone of the most strongly hypoxia-inducible proteins.

SUMMARY OF THE INVENTION

In one aspect, the instant invention concerns the identification ofMN/CA IX as one of the most strongly hypoxia-inducible proteins.Hypoxia-related MN/CA IX expression patterns indicate that it can serveas an intrinsic hypoxic marker, adding to the understanding of MN/CAIX's diagnostic and prognostic value.

Identified herein is the location of the HIF-1 consensus binding sitewithin the MN/CA 9 promoter shown in FIG. 6 (−506/+34) [SEQ ID NO: 27]and (−506/+43) [SEQ ID NO: 144] at the beginning of FIG. 1A. That HIF-1consensus binding site within the MN/CA 9 promoter is herein specifiedas beginning 3 by 5′ to the transcriptional start site, oriented on theantisense strand, reading 5′-TACGTGCA-3′ [SEQ ID NO: 145] shown in FIG.9 on the sense strand within the minimal promoter fragment (−36/+14)[SEQ ID NO: 146]. SEQ ID NO: 145 is also known as putative MN/CA 9hypoxia response element (HRE). [Wykoff et al., Cancer Res., 60:7075-7083 (Dec. 15, 2000).]

-   -   “Hypoxia-inducible factors (HIFs) locate to HIF-binding sites        (HBSs) within the hypoxia-response elements (HREs) of        oxygen-regulated genes . . . .    -   Limited O₂ supply (hypoxia) can alter the expression pattern of        a specific set of genes involved in mammalian O₂ homeostasis,        such as those encoding erythropoietin (EPO), transferrin or        vascular endothelial growth factor (VEGF).        [Camenisch et al., Pflügers Arch—Eur J Physiol, 443: 240-249 at        240 (2001).]

Camenisch et al. (2001) list in Table 1 at page 243 the HBSs for allgenes that had been identified as direct targets of HIF-1 functionincluding that for MN/CA IX and state at page 242: “The HIF-1 consensusDNA binding site contains CGTG [SEQ ID NO: 147] as the conserved coresequence, usually preceded by an adenosine and followed by a cytosineresidue.” Such a described “usual” conserved core sequence would thenread ACGTGC [SEQ ID NO: 148] which sequence is found in the HBS forMN/CA 9, which is specified in Camenisch et al. to be TACGTGCATT [SEQ IDNO: 149].

Musson et al. “Screening for Mutations in and around the HRE in thePromoter Region of the VEGF Gene in ALS Patients and Controls,” ALSSymp. Abstracts, pp. 62-63 (Abstract No. P23), 13^(th) InternationalSymposium on ALS/MND, Nov. 17-19, 2002, Melbourne, Australia (October2002) [Poster Theme 2: Genetics and Epidemiology] point out that thetranscription factor HIF-1 is known to bind to the consensus sequence 5′(G/CfT)-ACGTGC (G/T) [SEQ ID NO: 110] within the promoter of genes whichare up-regulated by HIF-1 during hypoxia. Musson et al. points out:

-   -   Hypoxia induction further requires the formation of a complex        between HIF-1 and other transcription factors bound to adjacent        sites . . . . In VEGF at least two sequences adjacent to the        HIF-1 binding site are essential for enhanced function in        hypoxia . . . . Together these sequences are known as the        hypoxia response element (HRE). An AP-1 site located further        downstream from the HIF-1 site has also been implicated in        hypoxic VEGF regulation.

Experiments described herein delineate the nature of the MN/CA 9 HRE.FIG. 6 shows some of the identified transcription factors within theMN/CA 9 promoter (SEQ ID NOS: 27 and 144), and other downstreamtranscription sites are herein disclosed which may be significant toenhancing hypoxia induction. Ones of skill in the art will recognizetranscription sites within the MN/CA 9 promoter and flanking regions inview of the detailed MN/CA 9 sequence information provided herein.

The MN/CA 9 HRE can be considered in one sense to comprise the HIF-1consensus binding site within the MN/CA 9 promoter preferably as shownabove to be SEQ ID NO: 145 and alternatively as SEQ ID NO: 149 as shownin Table 1 of Camenisch et al., supra. Variations in such HIF-1consensus binding sites can be visualized as maintaining or promotingthe hypoxia-inducible activity of the MN/CA 9 promoter. For example, oneof skill might visualize a nt sequence comprising the HIF-1 consensusbinding site CGTG [SEQ ID NO: 147] or as ACGTGC [SEQ ID NO: 148] or as5′(G/C/T-ACGTGC (G/T) [SEQ ID NO: 150], among other known HIF-1consensus binding sequences, as for example, those set forth in Table 1of Camenisch at al., supra.

In another sense, the MN/CA 9 HRE can be considered to comprise a HIF-1consensus binding site and flanking sequences, preferably immediatelyadjacent [see, e.g. the MN/CA 9 genomic sequence (SEQ ID NO: 5) shown inFIG. 2A-F] within which are located the binding sites of othertranscription factors with which HIF-1 could form a complex therebyenhancing hypoxia induction. Preferred candidates for the location ofthe MN/CA 9 HRE in the expanded sense of comprising additionaltranscription factor sites to which HIF-1 could complex include theMN/CA 9 promoter [SEQ ID NOS: 27 and 144] and fragments of said promoterthat comprise the HIF-1 consensus binding site, variations as describedabove, but preferably SEQ ID NOS: 145 and 149, more preferably SEQ IDNO: 145.

Exemplary and preferred MN/CA 9 promoter fragments include the MN5promoter fragment (−172/+31) [SEQ ID NO: 91], nearly identical to MN 5promoter fragment (−173/+31) [SEQ ID NO: 21], closely related promoterfragment (−173/+43) [SEQ ID NO: 151], MN4 promoter fragment (−243/+31)[SEQ ID NO: 93], MN6 promoter fragment (−58/+31) [SEQ ID NO: 94], MN7(−30/+31) [SEQ ID NO: 95], and a related minimal promoter fragment(−36/+14) [SEQ ID NO: 146]. Particularly preferred MN/CA 9 promoterfragments in the HRE sense include SEQ ID NOS: 21, 91, 94, 146 and 151.The determination of the complex of HIF-1 for the MN/CA 9 promoter willclarify the nature of the MN/CA 9 HRE in the expanded sense.

The particularly tight regulation of MN/CA 9 by hypoxia indicates thatits promoter [(−506/+34) SEQ ID NO: 27 and (−506/+43) SEQ ID NO: 144] orMN promoter fragments containing a MN/CA 9 HBS, wherein such MN/CA 9promoter fragments are exemplified by MN5 (−172/+31) [SEQ ID NO: 91],(−173/+31) [SEQ ID NO: 21], (−173/+43) [SEQ ID NO: 151], MN4 (−243/+31)[SEQ ID NO: 93], MN6 promoter fragment (−58/+31) [SEQ ID NO: 94], MN 7(−30/+31) [SEQ ID NO: 95], and the related minimal promoter (−36/+14)[SEQ ID NO: 146], among many other such MN/CA 9 promoter fragments,would be useful in target specific delivery systems of conditionallylethal drugs (such as enzyme converted prodrugs) in hypoxic cells. Asindicated above, particularly preferred MN/CA 9 promoter fragmentsinclude SEQ ID NOS: 21, 91, 94, 146 and 151, as well as related andvaried promoter fragments as indicated above. The MN/CA 9 promoter orMN/CA 9 promoter fragments comprising the HIF-1 consensus bindingsequence (varied as indicated above as long as the hypoxia inducibleactivity is maintained, and preferably enhanced) can be used to drivehypoxia inducibility in heterologous promoters.

Another aspect of this invention are therapeutic methods to inhibit thegrowth of vertebrate, preferably mammalian, more preferably human,preneoplastic or neoplastic cells in hypoxic regions of tumors, or ofcells in hypoxic conditions caused other than by cancer, preferably insuch cells expressing MN/CA IX at an abnormally high level. Such methodscomprise transfecting such a cell with a vector comprising a nucleicacid that encodes a cytotoxic protein/polypeptide, such as HSVtk,operatively linked to the MN gene promoter or a MN gene promoterfragment that comprises the HIF-1 consensus binding site as describedabove. Such a MN/CA 9 promoter fragment is preferably as described aboveand can comprise a nt sequence selected from the group consisting of,for example, SEQ ID NOS: 21, 91, 93, 94, 95, 146 and 151, and preferablythe nt sequence is selected from the group consisting of SEQ ID NOS: 21,91, 94, 146 and 151.

Such a therapeutic vector may also comprise a nucleic acid encoding acytokine, such as, IL-2 or IFN. A variety of vectors can be visualizedfor therapeutic purposes including retroviral vectors among many otherconstructs.

A further aspect of the instant invention concerns such vectorsthemselves that comprise a nucleic acid that encodes a cytotoxic proteinor cytotoxic polypeptide operatively linked to the MN gene promoter or aMN/CA 9 promoter fragment that comprises the HIF-1 consensus bindingsequence as described above, wherein said vector, when transfected intoa vertebrate preneoplastic or neoplastic cell or such a cell underhypoxic conditions caused other than by cancer, preferably such a cellexpressing MN/CA 9 at an abnormally high level, inhibits the growth ofsaid cell. In one preferred embodiment said cytotoxic protein is HSVthymidine kinase. Preferably, said vector further comprises a nucleicacid encoding a cytokine operatively linked to said MN gene promoter orMN/CA 9 promoter fragment. In alternative and preferred embodiments,said cytokine is interferon or interleukin-2.

More specifically, one aspect of the instant invention includes: Avector comprising a nucleic acid that encodes a cytotoxic protein orcytotoxic polypeptide operatively linked to a MN/CA 9 promoter or MN/CA9 promoter fragment which comprises a HIF-1 consensus binding sequence,wherein said vector, when transfected into a vertebrate cell thatabnormally expresses MN/CA IX protein, such as a preneoplastic orneoplastic cell, inhibits the growth of said cell, wherein said MN/CA 9gene promoter or MN/CA 9 gene promoter fragment has a nucleotidesequence selected from the group consisting of:

(a) SEQ ID NOS: 27 and 144;

(b) nucleotide sequences that are fully complementary to the nucleotidesequences of (a); and

(c) nucleotide sequences which specifically hybridize under stringenthybridization conditions of 50% formamide at 42° C. to any of thenucleotide sequences of (a) and (b). Exemplary and preferred MN/CA 9promoter fragments are set forth above.

MN/CA IX as a hypoxia marker is useful in making therapeutic decisions.For example, a cancer patient whose tumor is shown to express MN/CA IXat an abnormally high level would not be a candidate for certain kindsof chemotherapy and radiotherapy, but would be a candidate forhypoxia-selective chemotherapy.

Brown, J. M., “Exploiting the hypoxic cancer cell: mechanisms andtherapeutic strategies,” Molecular Medicine Today, 6: 157-162 (April2000) points out at page 157 that “solid tumours are considerably lesswell oxygenated than normal tissues. This leads to resistance toradiotherapy and anticancer chemotherapy, as well as predisposing toincreased tumour metastases.” Brown explains how tumor hypoxia can beexploited in cancer treatment.

One strategy to exploit tumor hypoxia for cancer treatment proposed byBrown, id. is to use drugs that are toxic only under hypoxic conditions.Exemplary and preferred drugs that could be used under that strategyinclude tirapazamine and AQ4N, a di-N-oxide analogue of mitozantrome.

A second mode of exploiting hypoxia proposed by Brown, id. is by genetherapy strategies developed to take advantage of the selectiveinduction of HIF-1. Brown notes that a tumor-specific delivery systemcan be developed wherein a promoter that is highly responsive to HIF-1would drive the expression of a conditionally lethal gene under hypoxicbut not normoxic conditions. “Expression of an enzyme not normally foundin the human body could, under the control of a hypoxia-responsivepromoter, convert a nontoxic pro-drug into a toxic drug in the tumour.”[Brown, id., page 160.] Exemplary is the use of the bacterial cytosinedeaminase, which converts the nontoxic 5-fluorocytosine to theanticancer drug 5-fluorouracil (5FU) cited by Brown to Trinh et al.,Cancer Res., 55: 4808-4812 (1995).

Ratcliffe et al., U.S. Pat. Nos. 5,942,434 and 6,265,390 explain howanti-cancer drugs become activated under hypoxia [Workman and Stafford,Cancer and Metastasis Reviews, 12: 73-82 (1993)], but that the use of adrug activation system, wherein the enzyme that activates the drug issignificantly increased under hypoxia, results in much enhancedtherapeutic effect. Ratcliffe et al., supra in the last five paragraphsin the Summary of the Invention states

-   -   The invention provides a nucleic acid construct comprising at        least one gene encoding a species having activity against        disease, operatively linked to a hypoxically inducible        expression control sequence.    -   When the construct is present in a suitable host cell,        expression of the gene will thus be regulated according to the        level of oxygenation. Preferably the expression control sequence        is a promoter or enhancer. In a host cell under hypoxic        conditions, expression of the gene will be initiated or        upregulated, while under conditions of normoxia (normal oxygen        level) the gene will be expressed at a lower level or not        expressed at all. The expression level may vary according to the        degree of hypoxia. Thus, a gene product which has therapeutic        activity can be targeted to cells affected by disease, eg.        tumour cells.    -   The species encoded by the gene in the construct according to        the invention may be for example a cytokine, such as        interleukin-2 (IL-2) which is known to be active in the immune        response against tumours. Genes encoding other molecules which        have an anti-tumour effect may also be used.    -   In a preferred embodiment of the construct according to the        invention, the species encoded by the gene is a pro-drug        activation system, for example the thymidine phosphorylase        enzyme, which converts a relatively inactive drug into a much        more potent one. Transfection of the thymidine phosphorylase        gene into human breast cancer cells has been shown to greatly        increase the sensitivity of the cancer cells to 5-deoxy-5FU . .        . . The thymidine phosphorylase gene has not previously been        reported as an agent for gene therapy. Another pro-drug        activation system which can be used is cytosine deaminase, which        activates the pro-drug 5-fluorocytosine (5-FC) to form the        antitumour agent 5-fluorouracil (5-FU). A further example of a        pro-drug activation system for use in the invention is        cytochrome p450 to activate the drug SR4233 (Walton et al,        [Biochem. Pharmacol. 44: 251-259] 1992).    -   The construct according to the invention may contain more than        one gene and more than one type of gene. Additional genes may        encode further species having activity against disease, or they        may have gene products with other activities.

In one aspect, the present invention provides diagnostic/prognostictools for determining the presence of hypoxia in a tissue in an animal,preferably a vertebrate, more preferably a mammal, still more preferablya human, and for measuring the relative degree of hypoxia in saidanimal.

In another aspect, the present invention provides tools for gene therapydesigned to exploit hypoxic conditions therapeutically.

In still another aspect, the present invention provides prognostic toolsfor patients with diseases associated with hypoxic conditions.

In one embodiment, the present invention provides for an expressionvector to determine the presence of hypoxia in a tissue in an animal. Inanother embodiment, the present invention provides for an expressionvector to determine the relative degree of hypoxia in a tissue of ananimal.

In one aspect, the invention is directed to the MN/CA 9 hypoxia-responseelement (HRE) and MN/CA9 promoter fragments comprising said HREincluding the MN/CA 9 HIF-1 consensus binding sequence or a variationthereof, preferably also comprising elements to enhance hypoxiainducibility. The MN/CA 9 HRE has several utilities. For example, theMN/CA 9 HRE or MN/CA 9 promoter fragments comprising said MN/CA 9 HRE ora fragment of said MN/CA 9 HRE, for example, at least the MN/CA 9 HIF-1consensus binding sequence (HBS), can be inserted into a suitableexpression vector, in combination with, preferably within, a promoter orpromoter fragment operatively linked to a gene, preferably a gene'scoding region. Cells can be transformed with such an expression vector,and the protein expressed therein will be regulated according to thedegree of oxygenation. Under hypoxia, gene expression will be initiatedor increased; under conditions of normoxia, gene expression will bereduced or eliminated.

This invention also concerns recombinant nucleic acid molecules thatcomprise a MN/CA 9 HRE or a MN/CA 9 promoter fragment comprising saidMN/CA 9 HRE or a MN/CA 9 HIF-1 consensus binding sequence. Saidrecombinant nucleic acid molecules may also comprise a nucleic acidsequence that encodes a non-MN/CA IX protein or polypeptide, and/or anon-MN/CA 9 HRE, a non-MN/CA 9 HBS, a non-MN/CA 9 promoter or promoterfragment, and one or more enhancer elements (that enhance hypoxiainducibility). Examples of a coding sequence for a non-MN/CA 9protein/polypeptide include the DNA sequence coding for the luciferasegene, the alpha-peptide coding region of beta-galactosidase, and asequence coding for glutathione S-transferase. Further, claimed hereinare such recombinant fusion proteins/polypeptides which aresubstantially pure and non-naturally occurring.

According to one aspect of the invention, a gene regulated by the MN/CA9 HRE, or by a MN/CA 9 promoter fragment containing a MN/CA 9 HRE orHBS, in the vector may encode for a cytokine, such as interleukin-2, orother molecules with known anti-tumor effects.

In a preferred embodiment, the gene regulated by the MN/CA 9 HRE or byMN/CA 9 promoter fragment comprising a MN/CA 9 HRE or HBS encodes for apro-drug activation system, such as the thymidine phosphorylase enzyme,which converts an inactive drug into an active one. Other pro-drugactivation systems according to the invention are cytosine deaminase,which activates the pro-drug 5-flyorocytosine (5-FC) to form theantitumor drug 5-fluorouracil (5-FU), and cytochrome p450 to activatethe drug SR4233.

Host cells transformed with the constructs of this invention are alsoencompassed within the scope of the invention.

Also disclosed herein are methods to use the MN/CA 9 gene and nucleicacid fragments thereof, including the herein described MN/CA 9 promoterand promoter fragments, particularly those comprising the MN/CA 9 HRE(preferably enhanced) and/or HIF-1 consensus binding sequence, MN/CA IXproteins/polypeptides, MN/CA IX-specific antibodies, whether monoclonal,polyclonal and/or antibody fragments, to identify hypoxic conditions,whether chronic or acute, particularly chronic, and/or to targettherapeutic drugs, including for example, enzyme activated pro-drugs,cytotoxic proteins/polypeptides, lethal drugs (preferably conditionallylethal that is, for example, lethal under hypoxic conditions, or onlyexpressed under hypoxic conditions) to hypoxic tissues or cells.

Further identified herein is the location of the MN protein bindingsite. Of particular importance is the region within theproteoglycan-like domain, aa 61-96 (SEQ ID NO: 97) which contains a6-fold tandem repeat of 6 amino acids, and within which the epitope forthe M75 MAb resides in at least two copies, and within which the MNbinding site is considered to be located. An alternative MN binding sitemay be located in the CA domain.

Also identified are MN proteins and MN polypeptides that compete forattachment to cells with immobilized MN protein. Such MNproteins/polypeptides prevent cell-cell adhesion and the formation ofintercellular contacts.

Disclosed herein are cell adhesion assay methods that are used toidentify binding site(s) on the MN protein to which vertebrate cells,preferably mammalian cells, more preferably human cells, bind. Such a MNbinding site is then identified as a therapeutic target which can beblocked with MN-specific antibodies, or inorganic or organic molecules,preferably organic molecules, more preferably proteins/polypeptides thatspecifically bind to said site.

Further disclosed are therapeutic methods to treat patients withpreneoplastic/neoplastic disease associated with or characterized byabnormal MN expression, which methods are based on blocking said MNbinding site with molecules, inorganic or organic, but preferablyorganic molecules, more preferably proteins/polypeptides, that bindspecifically to said binding site. The growth of a vertebratepreneoplastic/neoplastic cell that abnormally expresses MN protein canbe inhibited by administering such organic or inorganic molecules,preferably organic molecules, more preferably proteins/polypeptides in atherapeutically effective amount in a physiologically acceptableformulation. Such a preferred therapeutic protein/polypeptide is hereinconsidered to comprise an amino acid sequence selected from the groupconsisting of SEQ ID NOS: 107-109. Such heptapeptides are considered tobe comprised by MN protein partner(s). Blocking the interaction betweenMN protein and its binding partner(s), is expected to lead to a decreaseof tumor growth.

Further provided are other therapeutic methods wherein the growth of avertebrate, preferably mammalian, more preferably human, preneoplasticor neoplastic cell that abnormally expresses MN protein is inhibited.Said methods comprise transfecting said cell with a vector comprising anexpression control sequence operatively linked to a nucleic acidencoding the variable domains of an MN-specific antibody, wherein saiddomains are separated by a flexible linker peptide, preferably SEQ IDNO: 116. Preferably said expression control sequence comprises the MNgene promoter or a MN/CA 9 promoter fragment comprising a HIF-1consensus sequence as described above.

Aspects of the instant invention disclosed herein are described in moredetail as follows. The therapeutic use of organic or inorganicmolecules, preferably organic molecules, is disclosed. Preferred suchmolecules bind specifically to a site on MN protein to which vertebratecells adhere in a cell adhesion assay, wherein said molecule when testedin vitro inhibits the adhesion of cells to MN protein. Further preferredare such molecules, which when in contact with a vertebratepreneoplastic or neoplastic cell that abnormally expresses MN protein,inhibit the growth of said cell. Said vertebrate cells are preferablymammalian and more preferably human.

Preferably such a molecule is organic, and more preferably such aorganic molecule is a protein or a polypeptide. Still furtherpreferably, said protein or polypeptide comprises an amino acid sequenceselected from the group consisting of SEQ ID NOS: 107, 108, 109, 137 and138. Even more preferably, said polypeptide is selected from the groupconsisting of SEQ ID NOS: 107, 108, 109, 137 and 138.

The site on MN proteins to which vertebrate cells adhere in said celladhesion assay is preferably within the proteoglycan-like domain [SEQ IDNO: 50] or within the carbonic anhydrase domain [SEQ ID NO: 51] of theMN protein. Preferably that site comprises an amino acid sequenceselected from the group consisting of SEQ ID NOS: 10 and 97-106. Stillfurther preferably, that site has an amino acid sequence selected fromthe group consisting of SEQ ID NOS: 10 and 97-106.

Another aspect of this invention concerns MN proteins and MNpolypeptides which mediate attachment of vertebrate cells in a celladhesion assay, wherein said MN protein or MN polypeptide whenintroduced into the extracellular fluid environment of vertebrate cellsprevents the formation of intercellular contacts and the adhesion ofsaid vertebrate cells to each other. Such MN proteins and MNpolypeptides may be useful to inhibit the growth of vertebratepreneoplastic or neoplastic cells that abnormally express MN protein,when such MN proteins or MN polypeptides are introduced into theextracellular fluid environment of such vertebrate cells. Saidvertebrate cells are preferably mammalian, and more preferably human.

Said MN proteins or MN polypeptides which mediate attachment ofvertebrate cells in a cell adhesion assay, preferably have amino acidsequences from SEQ ID NO: 97, from SEQ ID NO: 50, or from SEQ ID NO: 51,more preferably from SEQ ID NO: 50. Still more preferably such MNproteins or MN polypeptides comprise amino acid sequences selected fromthe group consisting of SEQ ID NOS: 10 and 97-106. Alternatively, saidMN polypeptides are selected from the group consisting of SEQ ID NOS: 10and 97-106.

Representative MN proteins and MN polypeptides which mediate attachmentof vertebrate cells in a cell adhesion assay, are specifically bound byeither the M75 monoclonal antibody that is secreted from the hybridomaVU-M75, which was deposited at the American Type Culture Collectionunder ATCC No. HB 11128, or by the MN12 monoclonal antibody that issecreted from the hybridoma MN 12.2.2, which was deposited at theAmerican Type Culture Collection under ATCC No. HB 11647, or by bothsaid monoclonal antibodies.

Another aspect of the instant invention is a method of identifying asite on an MN protein to which vertebrate cells adhere by testing aseries of overlapping polypeptides from said MN protein in a celladhesion assay with vertebrate cells, and determining that if cellsadhere to a polypeptide from said series, that said polypeptidecomprises a site on said MN protein to which vertebrate cells adhere.

Still another aspect of the instant invention is a vector comprising anexpression control sequence operatively linked to a nucleic acidencoding the variable domains of a MN-specific antibody, wherein saiddomains are separated by a flexible linker polypeptide, and wherein saidvector, when transfected into a vertebrate preneoplastic or neoplasticcell that abnormally expresses MN protein, inhibits the growth of saidcell. Preferably said expression control sequence comprises the MN genepromoter or a MN/CA 9 promoter fragment, preferably comprising the HIF-1consensus binding sequence as described above, operatively linked tosaid nucleic acid. Further preferably, said flexible linker polypeptidehas the amino acid sequence of SEQ ID NO: 116, and even furtherpreferably, said MN gene promoter has the nucleotide sequence of SEQ IDNO: 27.

The MN gene promoter is characterized herein. The identification of thebinding site for a repressor of MN transcription is disclosed.Mutational analysis indicated that the direct repeat AGGGCacAGGGC [SEQID NO: 143] is required for efficient repressor binding.

Identification of the protein that binds to the repressor andmodification of its binding properties is another route to modulate MNexpression leading to cancer therapies. Suppression of MN expression intumor cells by overexpression of a negative regulator is expected tolead to a decrease of tumor growth. A repressor complex comprising atleast two subunits was found to bind to SEQ ID NO: 115 of the MN genepromoter. A repressor complex, found to be in direct contact with SEQ IDNO: 115 by UV crosslinking, comprised two proteins having molecularweights of 35 and 42 kilodaltons, respectively.

ABBREVIATIONS

The following abbreviations are used herein:

5-FC 5-flyorocytosine 5-FU 5-fluorouracil aa amino acid ATCC AmericanType Culture Collection bp base pairs BLV bovine leukemia virus BSAbovine serum albumin BRL Bethesda Research Laboratories CA carbonicanhydrase CAM cell adhesion molecule CARP carbonic anhydrase relatedprotein CAT chloramphenicol acetyltransferase Ci curie cm centimeter CMVcytomegalovirus cpm counts per minute C-terminis carboxyl-terminus CTLcytotoxic T lymphocytes ° C. degrees centigrade DEAE diethylaminoethylDMEM Dulbecco modified Eagle medium ds double-stranded EDTAethylenediaminetetraacetate EGF epidermal growth factor EIA enzymeimmunoassay ELISA enzyme-linked immunosorbent assay EMSA electrophoreticmobility shift assay EPO erythropoietin F fibroblasts FACScytofluorometric study FCS fetal calf serum FITC fluoresceinisothiocyanate FTP DNase 1 footprinting analysis GST-MN fusion proteinMN glutathione S-transferase GVC ganciclovir H HeLa cells HBSHIF-binding site H-E haematoxylin-eosin HEF human embryo fibroblastsHeLa K standard type of HeLa cells HeLa S Stanbridge's mutant HeLaD98/AH.2 H/F-T hybrid HeLa fibroblast cells that are tumorigenic;derived from HeLa D98/AH.2 H/F-N hybrid HeLa fibroblast cells that arenontumorigenic; derived from HeLa D98/AH.2 HIF hypoxia-inducible factorHPV Human papilloma virus HRE hypoxia response element HRP horseradishperoxidase HSV Herpes simplex virus IC intracellular IFN interferon IL-2interleukin-2 Inr initiator IPTG isopropyl-beta-D-thiogalacto-pyranosidekb kilobase kbp kilobase pairs kd or kDa kilodaltons KS keratan sulphateLCMV lymphocytic choriomeningitis virus LTR long terminal repeat M molarmA milliampere MAb monoclonal antibody MCSF macrophage colonystimulating factor ME mercaptoethanol MEM minimal essential medium min.minute(s) mg milligram ml milliliter mM millimolar MMC mitomycin C mmolmillimole MLV murine leukemia virus N normal concentration NEG negativeng nanogram nm nanometer nt nucleotide N-terminus amino-terminus ODNoligodeoxynucleotide ORF open reading frame PA Protein A PBS phosphatebuffered saline PCR polymerase chain reaction PEST combination ofone-letter abbreviations for proline, glutamic acid, serine, threoninePG proteoglycan PGK phosphoglycerate kinase pI isoelectric point PMAphorbol 12-myristate 13-acetate POS positive Py pyrimidine RACE rapidamplification of cDNA ends RCC renal cell carcinoma RIA radioimmunoassayRIP radioimmunoprecipitation RIPA radioimmunoprecipitation assay RNPRNase protection assay RT-PCT reverse transcription polymerase chainreaction SAC Staphylococcus aureus cells S. aureus Staphylococcus aureussc subcutaneous SDRE serum dose response element SDS sodium dodecylsulfate SDS-PAGE sodium dodecyl sulfate-polyacrylamide gelelectrophoresis SINE short interspersed repeated sequence SP signalpeptide SP-RIA solid-phase radioimmunoassay SSDS synthetic splice donorsite SSH subtractive suppressive PCR SSPE NaCl (0.18 M), sodiumphosphate (0.01 M), EDTA (0.001 M) SV40 simian virus 40 TBETris-borate/EDTA electrophoresis buffer TC tissue culture TCAtrichloroacetic acid TC media tissue culture media TC tissue culture tkthymidine kinase TM transmembrane TMB tetramethylbenzidine Tris tris(hydroxymethyl) aminomethane μCi microcurie μg microgram μl microliterμM micromolar VEGF vascular endothelial growth factor VSV vesicularstomatitis virus VV vaccinia virus X-MLV xenotropic murine leukemiavirus

Cell Lines

-   AGS—cell line derived from a primary adenogastric carcinoma    [Barranco and Townsend, Cancer Res., 43: 1703 (1983) and Invest. New    Drugs, 1: 117 (1983)]; available from the ATCC under CRL-1739;-   BL-3—bovine B lymphocytes [ATCC CRL-8037; leukemia cell    suspension; J. Natl. Cancer Inst. (Bethesda) 40: 737 (1968)];-   C33—a cell line derived from a human cervical carcinoma biopsy    [Auersperg, N., J. Nat'l. Cancer Inst. (Bethesda), 32: 135-148    (1964)]; available from the ATCC under HTB-31;-   C33A—human cervical carcinoma cells [ATCC HTB-31; J. Natl. Cancer    Inst. (Bethesda) 32: 135 (1964)];-   C4.5—CHO wild-type, parental to Ka13, the same cell line as that    described in Wood et al., J. Biol. Chem. 273: 8360-8368 (1998);-   Cos—simian cell line [Gluzman, Y., Cell, 23: 175 (1981)];-   HeLa—from American Type Culture Collection (ATCC)-   HeLa K—standard type of HeLa cells; aneuploid, epithelial-like cell    line isolated from a human cervical adenocarcinoma [Gey et al.,    Cancer Res., 12: 264 (1952); Jones et al., Obstet. Gynecol., 38:    945-949 (1971)] obtained from Professor B. Korych, [Institute of    Medical Microbiology and Immunology, Charles University; Prague,    Czech Republic];-   HeLa—Mutant HeLa clone that is hypoxanthine D98/AH.2 guanine    phosphoribosyl transferase-deficient (HGPRT⁻) (also HeLa s) kindly    provided by Eric J. Stanbridge [Department of Microbiology, College    of Medicine, University of California, Irvine, Calif. (USA)] and    reported in Stanbridge et al., Science, 215: 252-259 (15 Jan. 1982);    parent of hybrid cells H/F-N and H/F-T, also obtained from E. J.    Stanbridge;-   Ka13—CHO mutant cell functionally defective for the HIF-1α subunit,    the same cell line as that described in Wood et al. (1998), supra;-   KATO III—cell line prepared from a metastatic form of a gastric    carcinoma [Sekiguichi et al., Japan J. Exp. Med., 48: 61 (1978)];    available from the ATCC under HTB-103;-   NIH-3T3—murine fibroblast cell line reported in Aaronson, Science,    237: 178 (1987);-   QT35—quail fibrosarcoma cells [ECACC: 93120832; Cell, 11: 95    (1977)];-   Raj—human Burkitt's lymphoma cell line [ATCC CCL-86; Lancet. 1: 238    (1964)];-   Rat2TK⁻—cell line (rat embryo, thymidine kinase mutant) was derived    from a subclone of a 5′-bromo-deoxyuridine resistant strain of the    Fischer rat fibroblast 3T3-like cell line Rat1; the cells lack    appreciable levels of nuclear thymidine kinase [Ahrens, B.,    Virology, 113: 408 (1981)];-   SiHa—human cervical squamous carcinoma cell line [ATCC HTB-35;    Friedl et al., Proc. Soc. Exp. Biol. Med., 135: 543 (1990)];-   XC—cells derived from a rat rhabdomyosarcoma induced with Rous    sarcoma virus-induced rat sarcoma [Svoboda, J., Natl. Cancer Center    Institute Monograph No. 17. IN: “International Conference on Avian    Tumor Viruses” (J. W. Beard ed.), pp. 277-298 (1964)], kindly    provided by Jan Svoboda [Institute of Molecular Genetics,    Czechoslovak Academy of Sciences; Prague, Czech Republic]; and-   CGL1—H/F-N hybrid cells (HeLa D98/AH.2 derivative);-   CGL2—H/F-N hybrid cells (HeLa D98/AH.2 derivative);-   CGL3—H/F-T hybrid cells (HeLa D98/AH.2 derivative);-   CGL4—H/F-T hybrid cells (HeLa D98/Ah.2 derivative).

Nucleotide and Amino Acid Sequence Symbols

The following symbols are used to represent nucleotides herein:

Base Symbol Meaning A adenine C cytosine G guanine T thymine U uracil Iinosine M A or C R A or G W A or T/U S C or G Y C or T/U K G or T/U V Aor C or G H A or C or T/U D A or G or T/U B C or G or T/U N/X A or C orG or T/U

There are twenty main amino acids, each of which is specified by adifferent arrangement of three adjacent nucleotides (triplet code orcodon), and which are linked together in a specific order to form acharacteristic protein. A three-letter or one-letter convention is usedherein to identify said amino acids, as, for example, in FIG. 1 asfollows:

3 Ltr. 1 Ltr. Amino acid name Abbrev. Abbrev. Alanine Ala A Arginine ArgR Asparagine Asn N Aspartic Acid Asp D Cysteine Cys C Glutamic Acid GluE Glutamine Gln Q Glycine Gly G Histidine His H Isoleucine Ile I LeucineLeu L Lysine Lys K Methionine Met M Phenylalanine Phe F Proline Pro PSerine Ser S Threonine Thr T Tryptophan Trp W Tyrosine Tyr Y Valine ValV Unknown or other X

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A-C provides the nucleotide sequence for a MN cDNA [SEQ ID NO: 1]clone isolated as described herein. FIG. 1A-C also sets forth thepredicted amino acid sequence [SEQ ID NO: 2] encoded by the cDNA.

FIG. 2A-F provides a 10,898 bp complete genomic sequence of MN [SEQ IDNO: 5]. The base count is as follows: 2654 A; 2739 C; 2645 G; and 2859T. The 11 exons are in general shown in capital letters, but exon 1 isconsidered to begin at position 3507 as determined by RNase protectionassay.

FIG. 3 is a restriction map of the full-length MN cDNA. The open readingframe is shown as an open box. The thick lines below the restriction mapillustrate the sizes and positions of two overlapping cDNA clones. Thehorizontal arrows indicate the positions of primers R1 [SEQ ID NO: 7]and R2 [SEQ ID NO: 8] used for the 5′ end RACE. Relevant restrictionsites are BamHI (B), EcoRV (V), EcoRI (E), PstI (Ps), PvuII (Pv).

FIG. 4 schematically represents the 5′ MN genomic region of a MN genomicclone wherein the numbering corresponds to transcription initiationsites estimated by RACE.

FIG. 5 provides an exon-intron map of the human MN/CA IX gene. Thepositions and sizes of the exons (numbered, cross-hatched boxes), Alurepeat elements (open boxes) and an LTR-related sequence (firstunnumbered stippled box) are adjusted to the indicated scale. The exonscorresponding to individual MN/CA IX protein domains are separated bydashed lines designated PG (proteoglycan-like domain), CA (carbonicanhydrase domain), TM (transmembrane anchor) and IC (intracytoplasmictail). Below the map, the alignment of amino acid sequences illustratesthe extent of homology between the MN/CA IX protein PG region (aa53-111) [SEQ ID NO: 50] and the human aggrecan (aa 781-839) [SEQ ID NO:54].

FIG. 6 is a nucleotide sequence for the proposed promoter of the humanMN gene [SEQ ID NO: 27]. The nucleotides are numbered from thetranscription initiation site according to RNase protection assay.Potential regulatory elements are overlined. Transcription start sitesare indicated by asterisks (RNase protection) and dots (RACE) above thecorresponding nucleotides. The sequence of the 1st exon begins under theasterisks. FTP analysis of the MN4 promoter fragment revealed 5 regions(I-V) protected at both the coding and noncoding strands, and tworegions (VI and VII) protected at the coding strand but not at thenoncoding strand.

FIG. 7 provides a schematic of the alignment of MN genomic clonesaccording to their position related to the transcription initiationsite. All the genomic fragments except Bd3 were isolated from a lambdaFIX II genomic library derived from HeLa cells. Clone Bd3 was derivedfrom a human fetal brain library.

FIG. 8 schematically represents the MN protein structure. Theabbreviations are the same as used in FIG. 5. The scale indicates thenumber of amino acids.

FIG. 9 describes the functional analysis of human MN/CA 9 5′-flankingsequences in transient expression assays. Left panel, schematic diagramof reporter genes; the indicated MN/CA 9 wild-type and mutant sequenceswere inserted 5′ to a promoterless luciferase reporter gene. Arrow, 5′transcriptional initiation site. Underlined sequence, the MN/CA 9 HIF-1consensus binding sequence [SEQ ID NO: 145] within the MN/CA 9 HRE orconsidered to be the putative MN/CA 9 HRE [Wykoff et al., Cancer Res.,60: 7075-7083 (Dec. 15, 2000], whereas MN/CA 9 promoter fragments maycomprise enhancer elements with which the HIF-1 transcription factor cancomplex. Right panels, reporter gene activities in transientlytransfected cells. The MN/CA 9 promoter sequences are indicated to theleft of each column. SV-40, control minimal SV-40 promoter. A,activities in normoxic and hypoxic HeLa cells. B and C, activities inwild-type CHO (C4.5) cells (columns 1) and HIF-1 α-deficient CHO (Ka13)cells (columns 2). A, hypoxia-inducible activity of the MN/CA 9promoter. B. hypoxia-inducible activity of the MN/CA 9 promoter isablated in Ka13 cells. Cotransfection of HIF-1α restores induction byhypoxia in Ka13 cells and augments MN/CA 9 promoter activity in bothwild-type and Ka13 cells. In comparison, minimal effects are seen on theSV40 promoter. C, a minimal MN/CA 9 promoter (SEQ ID NO: 146] retainsHIF-1α-dependent, hypoxia inducible activity. Two mutations within theputative MN/CA 9 HRE or MN/CA 9 HIF-1 consensus binding sequence, MUT1and MUT2, completely ablate hypoxia-inducible activity, whereas basaltranscription is preserved. Columns, mean luciferase activitiescorrected for transfection efficiency from a typical experimentperformed in duplicate. Each duplicate experiment was repeated two tosix times. Numbers to the right are the ratios of hypoxic to normoxicexpression of the indicated reporter construct. Transfected cells wereincubated at 20% O₂ for 8 h and then incubated at 20% O₂ (normoxia) or0.1% O₂ (hypoxia) for 16 h. [FIG. 2 of Wykoff et al., Cancer Res., 60:7075-7083 (Dec. 15, 2000).]

DETAILED DESCRIPTION

The following references provide updated information concerning theMN/CA 9 gene and the MN/CA IX protein, which references are specificallyincorporated by reference herein as well as references cited therein andare useful to clarify any inconsistent details concerning the MN geneand protein:

-   Barto{hacek over (s)}ová et al., “Expression of carbonic anhydrase    IX in breast is associated with malignant tissues and is related to    overexpression of c-erbB2,” Journal of Pathology, 197: 314-321    (2002)-   Beasley et al., “Carbonic anhydrase IX, an endogenous hypoxia    marker, expression in head and neck squamous cell carcinoma and its    relationship to hypoxia, necrosis, and microvessel density,” Cancer    Res., 61(13): 5262-5267 (Jul. 1, 2001)-   Chia et al., “Prognostic Significance of a Novel Hypoxia Regulated    Marker, Carbonic Anhydrase IX (MN/CA IX), in Invasive Breast    Cancer,” Breast Cancer Research and Treatment, 64(1): pp. 43 (2000)-   Chia et al., “Prognostic significance of a novel hypoxia-regulated    marker, carbonic anhydrase IX, in invasive breast carcinoma,” J.    Clin. Oncol., 19(16): 3660-3668 (Aug. 15, 2001)-   Giatromanolaki et al., “Expression of Hypoxia-inducible Carbonic    Anhydrase-9 Relates to Angiogenic Pathways and Independently to Poor    Outcome in Non-Small Cell Lung Cancer,” Cancer Research, 61(21):    7992-7998 (Nov. 1, 2001)-   Ivanov et al., “Expression of Hypoxia-Inducible Cell-Surface    Transmembrane Carbonic Anhydrases in Human Cancer,” American Journal    of Pathology, 158(3): 905-919 (March 2001)-   Kaluz et al., “Transcriptional Regulation of the MN/CA9 Gene Coding    for the Tumor-associated Carbonic Anhydrase IX,” The Journal of    Biological Chemistry, 274(46): 32588-32595 (Nov. 12, 1999)-   Kaluzova et al., “P53 tumour suppressor modulates transcription of    the TATA-less gene coding for the tumour-associated carbonic    anhydrase MN/CA IX in MaTu Cells,” Biochemica et Biophysica Acta,    1491: 20-26 (2000)-   Kaluzova et al., “Characterization of the MN/CA 9 promoter proximal    region: a role for specificity protein (SP) and activator protein 1    (API) factors,” Biochemical Journal, 359(Pt 3): 669-677 (Nov. 1,    2001)-   Kivela et al., “Expression of transmembrane carbonic anhydrase    isoenzymes IX and XII in normal human pancreas and pancreatic    tumours,” Histochemistry and Cell Biology, 114(3): 197-204 (2000)-   Koukourakis et al., “Hypoxia-regulated Carbonic Anhydrase-9 (CA9)    Relates to Poor Vascularization and Resistance of Squamous Cell Head    and Neck Cancer to Chemoradiotherapy,” Clinical Cancer Research,    7(11): 3399-3403 (November 2001)-   Lieskovska et al., “Up-regulation of p53 by antisense expression of    HPV18 E6 oncogene does not influence the level of MN/CA IX    tumor-associated protein in HeLa cervical carcinoma cells,”    International Journal of Oncology, 13: 1081-1086 (1998)-   Lieskovska et al., “Study of in vitro conditions modulating    expression of MN/CA IX protein in human cell lines derived from    cervical carcinoma,” Neoplasma, 46: 17-24 (1999)-   Loncaster et al., “Carbonic Anhydrase (CAIX) Expression, a Potential    New Intrinsic Marker of Hypoxia: Correlations with Tumor Oxygen    Measurements and Prognosis in Locally Advanced Carcinoma of the    Cervix,” Cancer Res, 61(17): 6394-6399 (Sep. 1, 2001)-   Ortova Gut et al., “Gastric Hyperplasia in Mice With Targeted    Disruption of the Carbonic Anhydrase Gene Car9,” Gastroenterology,    123: 1889-1903 (2002)-   Parkkila et al., “Carbonic anhydrase inhibitor suppresses invasion    of renal cancer cells in vitro,” PNAS (USA), 97(5): 2220-2224 (Feb.    29, 2000)-   Pastorekova et al., “Carbonic anhydrase IX, a new player in a    HIF-directed orchestra implicated in cell adhesion,” Abstract    submitted to International Conference on Hypoxia/HIF Mediated    Responses in Tumor Biology, Univ. of Manchester, United Kingdom    (Nov. 27-29, 2002)-   Saarnio et al., “Immunohistochemistry of Carbonic Anhydrase Isozyme    IX (MN/CA IX) in Human Gut Reveals Polarized Expression in the    Epithelial Cells with the Highest Proliferative Capacity,” Journal    of Histochemistry & Cytochemistry, 46(4): 497-504 (1998)-   Saarnio et al., “Immunohistochemical Study of Colorectal Tumors for    Expression of a Novel Transmembrane Carbonic Anhydrase, MN/CA IX,    with Potential Value as a Marker of Cell Proliferation,” Am. J.    Pathol., 153(1): 279-285 (July 1998)-   Stouracova et al., “Preliminary crystallographic study of an    anti-MN/CA IX monoclonal antibody M75 Fab fragment complexed with    its epitope peptide,” Abstract submitted to 20th European    Crystallographic Meeting ECM 20 in Kraków (Aug. 25-31, 2001)-   Turner et al., “The hypoxia-inducible genes VEGF and CA9 are    differentially regulated in superficial vs invasive bladder cancer,”    British Journal of Cancer, 86: 1276-1282 (2002)-   Vermylen et al., “Carbonic anhydrase IX antigen differentiates    between preneoplastic malignant lesions in non-small cell lung    carcinoma,” Eur Respir J. 14: 806-811 (1999)-   Wykoff et al., “Hypoxia-inducible Expression of Tumor-associated    Carbonic Anhydrases,” Cancer Research, 60: 7075-7083 (Dec. 15, 2000)-   Wykoff et al., “Expression of the hypoxia-inducible and    tumor-associated carbonic anhydrases in ductal carcinoma in situ of    the breast,” Am. J. Pathol., 158(3): 1011-1019 (March 2001)-   Zavada et al., “Tumor-associated cell adhesion molecule MN/CA9:    Identification of the binding site,” Cancer Detection and    Prevention, 22 (Suppl. 1): 72 (Abstract #203) (1998)-   Zavada et al., “Biological Activity of MN/CA IX Protein: Inhibition    with Monoclonal Antibody or with Synthetic Oligopeptides,” Abstract    submitted to European Association of Cancer Research Meeting in    Halkidiki, Greece, May 30-Jun. 3, 2000-   Zavada et al., “Human tumour-associated cell adhesion protein MN/CA    IX: identification of M75 epitope and of the region mediating cell    adhesion,” British Journal of Cancer, 82(11): 1808-1813 (2000)-   Zavadova et al., “Two Functions of Tumor-Associated MN/CA IX    Protein,” Abstract submitted to European Association of Cancer    Research Meeting in Halkidiki, Greece, May 30-Jun. 3, 2000

MN/CA IX was first identified in HeLa cells, derived from humancarcinoma of cervix uteri, as both a plasma membrane and nuclear proteinwith an apparent molecular weight of 58 and 54 kilodaltons (kDa) asestimated by Western blotting. It is N-glycosylated with a single 3 kDacarbohydrate chain and under non-reducing conditions forms S-S-linkedoligomers [Pastorekova et al., Virology, 187: 620-626 (1992); Pastoreket al., Oncogene, 9: 2788-2888 (1994)]. MN/CA IX is a transmembraneprotein located at the cell surface, although in some cases it has beendetected in the nucleus [Zavada et al., Int. J. Cancer, 54: 268-274(1993); Pastorekova et al., supra].

MN is manifested in HeLa cells by a twin protein, p54/58N. Immunoblotsusing a monoclonal antibody reactive with p54/58N (MAb M75) revealed twobands at 54 kd and 58 kd. Those two bands may correspond to one type ofprotein that most probably differs by post-translational processing.Herein, the phrase “twin protein” indicates p54/58N.

Zavada et al., WO 93/18152 and/or WO 95/34650 disclose the MN cDNAsequence (SEQ ID NO: 1) shown herein in FIG. 1A-1C, the MN amino acidsequence (SEQ ID NO: 2) also shown in FIG. 1A-1C, and the MN genomicsequence (SEQ ID NO: 5) shown herein in FIG. 2A-2F. The MN gene isorganized into 11 exons and 10 introns.

The first thirty-seven amino acids of the MN protein shown in FIGS.1A-1C is the putative MN signal peptide [SEQ ID NO: 6]. The MN proteinhas an extracellular domain [amino acids (aa) 38-414 of FIGS. 1A-1C (SEQID NO: 87)], a transmembrane domain [aa 415-434 (SEQ ID NO: 52)] and anintracellular domain [aa 435-459 (SEQ ID NO: 53)]. The extracellulardomain contains the proteoglycan-like domain [aa 53-111 (SEQ ID NO: 50)]and the carbonic anhydrase (CA) domain [aa 135-391 (SEQ ID NO: 51].

Anticancer Drugs and Antibodies that Block Interaction of MN Protein andReceptor Molecules

MN protein is considered to be a uniquely suitable target for cancertherapy for a number of reasons including the following. (1) It islocalized on the cell surface, rendering it accessible. (2) It isexpressed in a high percentage of human carcinomas (e.g., uterinecervical, renal, colon, breast, esophageal, lung, head and neckcarcinomas, among others), but is not normally expressed to anysignificant extent in the normal tissues from which such carcinomasoriginate. (3) It is normally expressed only in the stomach mucosa andin some epithelia of the digestive tract (epithelium of gallbladder andsmall intestine). An anatomic barrier thereby exists between theMN-expressing preneoplastic/neoplastic and MN-expressing normal tissues.Drugs, including antibodies, can thus be administered which can reachtumors without interfering with MN-expressing normal tissues. (4) MAbM75 has a high affinity and specificity to MN protein. (5) MN cDNA andMN genomic clones which encompass the protein-coding and gene regulatorysequences have been isolated. (6) MN-specific antibodies have been shownto have among the highest tumor uptakes reported in clinical studieswith antitumor antibodies in solid tumors, as shown for the MN-specificchimeric antibody G250 in animal studies and in Phase I clinical trialswith renal carcinoma patients. (Steffens et al., J. Clin. Oncol., 15:1529 (1997).] Also, MN-specific antibodies have low uptake in normaltissues.

Data, e.g. as presented herein, are consistent with the following theoryconcerning how MN protein acts in normal tissues and inpreneoplastic/neoplastic tissues. In normal tissues (e.g., in stomachmucosa), MN protein is considered to be a differentiation factor. Itbinds with its normal receptor S (for stomach). Stomach carcinomas havebeen shown not to contain MN protein.

Ectopic expression of MN protein in other tissues causes malignantconversion of cells. Such ectopic expression is considered to be causedby the binding of MN protein with an alternative receptor H (for HeLacells), coupled to a signal transduction pathway leading to malignancy.Drugs or antibodies which block the binding site of MN protein forreceptor H would be expected to cause reversion ofprenoplastic/neoplastic cells to normal or induce their death.

Design and Development of MN-Blocking Drugs or Antibodies

A process to design and develop MN-blocking drugs, e.g., peptides withhigh affinity to MN protein, or antibodies, has several steps. First, isto test for the binding of MN protein to receptors based on the celladhesion assay described infra. That same procedure would also be usedto assay for drugs blocking the MN protein binding site. In view of thealternative receptors S and H, stomach epithelial cells or revertants(containing preferentially S receptors), HeLa cells (containing the Hreceptor and lacking the S receptor) would be used in the cell adhesionassay.

To identify the receptor binding site of MN protein, deletion variantsof MN protein lacking different domains can be used to identifyregion(s) responsible for interaction of MN protein with a receptor.Example 2 identifies and illustrates how to detect other binding siteson MN protein. A preferred MN binding site is considered to be closelyrelated or identical to the epitope for MAb M75, which is located in atleast 2 copies within the 6-fold tandem repeat of 6 amino acids [aa61-96 (SEQ ID NO: 97)] in the proteoglycan-like domain of the MNprotein. Smaller deletion variants can be prepared within that relevantdomain, e.g., fusion proteins with only small segments of MN protein canbe prepared. Also, controlled digestion of MN protein with specificproteases followed by separation of the products can be performed.

Further, peptides comprising the expected binding site can besynthesized. All of those products can be tested in cell adhesionassays, as exemplified below. [See, e.g., Pierschbacher and Ruoslahti,PNAS, 81:5985 (1984); Ruoslahti and Pierschbacher, Science, 238: 491.]

Molecules can be constructed to block the MN receptor binding site. Forexample, use of a phage display peptide library kit [as Ph.D®-7 Peptide7-Mer Library Kit from New England Biolabs; Beverly, Mass. (USA)] asexemplified in Examples 2 and 3, can be used to find peptides with highaffinity to the target molecules. Biologic activity of the identifiedpeptides will be tested in vitro by inhibition of cell adhesion to MNprotein, by effects on cell morphology and growth characteristics ofMN-related tumor cells (HeLa) and of control cells. [Symington, J. Biol.Chem., 267: 25744 (1992).] In vivo screening will be carried out in nudemice that have been injected with HeLa cells.

Peptides containing the binding site of the MN protein will be prepared[e.g. MAPs (multiple antigen peptides); Tam, J. P., PNAS (USA) 85: 5409(1988); Butz et al., Peptide Res., 7: 20 (1994)]. The MAPs will be usedto immunize animals to obtain antibodies (polyclonal and/or monoclonal)that recognize and block the binding site. [See, e.g., Brooks et al.,Cell, 79: 1157 (1994).] “Vaccination” would then be used to test forprotection in animals. Antibodies to the MN binding site couldpotentially be used to block MN protein's interaction(s) with othermolecules.

Computer modeling can also be used to design molecules with specificaffinity to MN protein that would mediate steric inhibition between MNprotein and its receptor. A computer model of the MN binding site forthe receptor will contain spatial, electrostatic, hydrophobic and othercharacteristics of this structure. Organic molecules complementary tothe structure, that best fit into the binding site, will be designed.Inorganic molecules can also be similarly tested that could block the MNbinding site.

The use of oncoproteins as targets for developing new cancertherapeutics is considered conventional by those of skill in the art.[See, e.g., Mendelsohn and Lippman, “Growth Factors,” pp. 114-133, IN:DeVita et al. (eds.), Cancer: Principles and Practice of Oncology(4^(th) Ed.; Lippincott; Philadelphia, 1993).] In its broadest sense,the design of blocking drugs can be based in competitive inhibitionexperiments. Such experiments have been used to invent drugs since thediscovery of sulfonamides (competitive inhibitors of para-aminobenzoicacid, a precursor of folic acid). Also, some cytostatics are competitiveinhibitors (e.g., halogenated pyrimidines, among others).

However, the application of such approaches to MN is new. In comparisonto other tumor-related molecules (e.g. growth factors and theirreceptors), MN has the unique property of being differentially expressedin preneoplastic/neoplastic and normal tissues, which are separated byan anatomic barrier.

MN Gene—Cloning and Sequencing

FIG. 1A-C provides the nucleotide sequence for a full-length MN cDNAclone isolated as described below [SEQ ID NO: 1]. FIG. 2A-F provides acomplete MN genomic sequence [SEQ ID NO: 5]. FIG. 6 shows the nucleotidesequence for a proposed MN promoter [SEQ ID NO: 27].

It is understood that because of the degeneracy of the genetic code,that is, that more than one codon will code for one amino acid [forexample, the codons TTA, TTG, CTT, CTC, CTA and CTG each code for theamino acid leucine (leu)], that variations of the nucleotide sequencesin, for example, SEQ ID NOS: 1 and 5 wherein one codon is substitutedfor another, would produce a substantially equivalent protein orpolypeptide according to this invention. All such variations in thenucleotide sequences of the MN cDNA and complementary nucleic acidsequences are included within the scope of this invention.

It is further understood that the nucleotide sequences herein describedand shown in FIGS. 1, 2 and 6, represent only the precise structures ofthe cDNA, genomic and promoter nucleotide sequences isolated anddescribed herein. It is expected that slightly modified nucleotidesequences will be found or can be modified by techniques known in theart to code for substantially similar or homologous MN proteins andpolypeptides, for example, those having similar epitopes, and suchnucleotide sequences and proteins/polypeptides are considered to beequivalents for the purpose of this invention. DNA or RNA havingequivalent codons is considered within the scope of the invention, asare synthetic nucleic acid sequences that encode proteins/polypeptideshomologous or substantially homologous to MN proteins/polypeptides, aswell as those nucleic acid sequences that would hybridize to saidexemplary sequences [SEQ. ID. NOS. 1, 5 and 27] under stringentconditions, or that, but for the degeneracy of the genetic code wouldhybridize to said cDNA nucleotide sequences under stringenthybridization conditions. Modifications and variations of nucleic acidsequences as indicated herein are considered to result in sequences thatare substantially the same as the exemplary MN sequences and fragmentsthereof.

Stringent hybridization conditions are considered herein to conform tostandard hybridization conditions understood in the art to be stringent.For example, it is generally understood that stringent conditionsencompass relatively low salt and/or high temperature conditions, suchas provided by 0.02 M to 0.15 M NaCl at temperatures of 50° C. to 70° C.Less stringent conditions, such as, 0.15 M to 0.9 M salt at temperaturesranging from 20° C. to 55° C. can be made more stringent by addingincreasing amounts of formamide, which serves to destabilize hybridduplexes as does increased temperature.

Exemplary stringent hybridization conditions are described in Sambrooket al., Molecular Cloning: A Laboratory Manual, pages 1.91 and 9.47-9.51(Second Edition, Cold Spring Harbor Laboratory Press; Cold SpringHarbor, N.Y.; 1989); Maniatis et al., Molecular Cloning: A LaboratoryManual, pages 387-389 (Cold Spring Harbor Laboratory; Cold SpringHarbor, N.Y.; 1982); Tsuchiya et al., Oral Surgery, Oral Medicine, OralPathology, 71(6): 721-725 (June 1991).

Zavada et al., WO 95/34650 described how a partial MN cDNA clone, afull-length MN cDNA clone and MN genomic clones were isolated andsequenced. Also, Zavada et al., Int. J. Cancer. 54: 268 (1993) describesthe isolation and sequencing of a partial MN cDNA of 1397 by in length.Briefly, attempts to isolate a full-length clone from the original cDNAlibrary failed. Therefore, the inventors performed a rapid amplificationof cDNA ends (RACE) using MN-specific primers, R1 and R2 [SEQ ID NOS: 7and 8], derived from the 5′ region of the original cDNA clone. The RACEproduct was inserted into pBluescript, and the entire population ofrecombinant plasmids was sequenced with an MN-specific primer ODN1 [SEQID NO: 3]. In that way, a reliable sequence at the very 5′ end of the MNcDNA as shown in FIG. 1 [SEQ ID NO: 1] was obtained.

Specifically, RACE was performed using 5′ RACE System [GIBCO BRL;Gaithersburg, Md. (USA)] as follows. 1 μg of mRNA (the same as above)was used as a template for the first strand cDNA synthesis which wasprimed by the MN specific antisense oligonucleotide, R1(5′-TGGGGTTCTTGAGGATCTCCAGGAG-3′) [SEQ ID NO: 7]. The first strandproduct was precipitated twice in the presence of ammonium acetate and ahomopolymeric C tail was attached to its 3′ end by TdT. Tailed cDNA wasthen amplified by PCR using a nested primer, R2(5′-CTCTAACTTCAGGGAGCCCTCTTCTT-3′) [SEQ ID NO: 8] and an anchor primerthat anneals to the homopolymeric tail(5′-CUACUACUACUAGGCCACGCGTCGACTAGTACGGGIIGGGIIGGGIIG-3′) [SEQ ID NO: 9].The amplified product was digested with BamHI and SalI restrictionenzymes and cloned into pBluescript II KS plasmid. After transformation,plasmid DNA was purified from the whole population of transformed cellsand used as a template for sequencing with the MN-specific primer ODN1[SEQ ID NO: 3; a 29-mer 5′ CGCCCAGTGGGTCATCTTCCCCAGAAGAG-3′].

To study MN regulation, MN genomic clones were isolated. One MN genomicclone (Bd3) was isolated from a human cosmid library prepared from fetalbrain using both MN cDNA as a probe and the MN-specific primers derivedfrom the 5′ end of the cDNA ODN1 [SEQ ID NO: 3, supra] and ODN2 [SEQ. IDNO.: 4; 19-mer (5′ GGAATCCTCCTGCATCCGG 3′)]. Sequence analysis revealedthat that genomic clone covered a region upstream from a MNtranscription start site and ending with the BamHI restriction sitelocalized inside the MN cDNA. Other MN genomic clones can be similarlyisolated.

FIG. 7 provides a schematic of the alignment of MN genomic clonesaccording to the transcription initiation site. Plasmids containing theA4a clone and the XE1 and XE3 subclones were deposited at the AmericanType Culture Collection (ATCC) on Jun. 6, 1995, respectively under ATCCDeposit Nos. 97199, 97200, and 97198.

Exon-Intron Structure of Complete MN Genomic Region

The complete sequence of the overlapping clones contains 10,898 bp (SEQID NO: 5). FIG. 5 depicts the organization of the human MN gene, showingthe location of all 11 exons as well as the 2 upstream and 6 intronicAlu repeat elements. All the exons are small, ranging from 27 to 191 bp,with the exception of the first exon which is 445 bp. The intron sizesrange from 89 to 1400 bp. The CA domain is encoded by exons 2-8, whilethe exons 1, 10 and 11 correspond respectively to the proteoglycan-likedomain, the transmembrane anchor and cytoplasmic tail of the MN/CA IXprotein. Table 1 below lists the splice donor and acceptor sequencesthat conform to consensus splice sequences including the AG-GT motif[Mount, Nucleic Acids Res. 10: 459-472 (1982)].

TABLE 1 Exon-Intron Structure of the Human MN Gene SEQ SEQ Genomic ID5′splice ID Exon Size Position** NO donor NO  1 445 *3507-3951 28AGAAG gtaagt 67  2 30  5126-5155 29 TGGAG gtgaga 68  3 171  5349-5519 30CAGTC gtgagg 69  4 143  5651-5793 31 CCGAG gtgagc 70  5 93  5883-5975 32TGGAG gtacca 71  6 67  7376-7442 33 GGAAG gtcagt 72  7 158  8777-8934 34AGCAG gtgggc 73  8 145  9447-9591 35 GCCAG gtacag 74  9 27  9706-9732 36TGCTG gtgagt 75 10 82 10350-70431 37 CACAG gtatta 76 11 191 10562-1043138 ATAAT end SEQ SEQ Genomic ID 3′splice ID Intron Size Position** NOacceptor NO  1 1174  3952-5125 39 atacag GGGAT 77  2 193  5156-5348 40ccccag GCGAC 78  3 131  5520-5650 41 acgcag TGCAA 79  4 89  5794-5882 42tttcag ATCCA 80  5 1400  5976-7375 43 ccccag GAGGG 81  6 1334  7443-877644 tcacag GCTCA 82  7 512  8935-9446 45 ccctag CTCCA 83  8 114 9592-9705 46 ctccag TCCAG 84  9 617  9733-10349 47 tcgcag GTGACA 85 10130 10432-10561 48 acacag AAGGG 86 **positions are related to ntnumbering in whole genomic sequence including the 5′ flanking region[FIG. 2A-F] *number corresponds to transcription initiation sitedetermined below by RNase protection assayMapping of MN Gene Transcription Initiation and Termination Sites

Zavada et al., WO 95/34650 describes the process of mapping the MN genetranscription initiation and termination sites. A RNase protection assaywas used for fine mapping of the 5′ end of the MN gene. The probe was auniformly labeled 470 nucleotide copy RNA (nt −205 to +265) [SEQ ID NO:55], which was hybridized to total RNA from MN-expressing HeLa and CGL3cells and analyzed on a sequencing gel. That analysis has shown that theMN gene transcription initiates at multiple sites, the 5′ end of thelongest MN transcript being 30 nt longer than that previouslycharacterized by RACE.

Characterization of the 5′ Flanking Region

The Bd3 genomic clone isolated from human fetal brain cosmid library wasfound to cover a region of 3.5 kb upstream from the transcription startsite of the MN gene. It contains no significant coding region. Two Alurepeats are situated at positions −2587 to −2296 [SEQ ID NO: 56] and−1138 to −877 [SEQ ID NO: 57] (with respect to the transcription startdetermined by RNP).

Nucleotide sequence analysis of the DNA 5′ to the transcription start(from nt −507) revealed no recognizable TATA box within the expecteddistance from the beginning of the first exon. However, the presence ofpotential binding sites for transcription factors suggests that thisregion might contain a promoter for the MN gene. There are severalconsensus sequences for transcription factors AP1 and AP2 as well as forother regulatory elements, including a p53 binding site [Locker andBuzard, J., DNA Sequencing and Mapping, 1: 3-11 (1990); Imagawa et al.Cell, 51: 251-260 (1987); El Deiry et al., Nat. Genet., 1: 44-49(1992)]. Although the putative promoter region contains 59.3% C+G, itdoes not have additional attributes of CpG-rich islands that are typicalfor TATA-less promoters of housekeeping genes [Bird, Nature, 321:209-213 (1986)]. Another class of genes lacking TATA box utilizes theinitiator (Inr) element as a promoter. Many of these genes are notconstitutively active, but they are rather regulated duringdifferentiation or development. The Inr has a consensus sequence ofPyPyPyCAPyPyPyPyPy [SEQ ID NO: 23] and encompasses the transcriptionstart site [Smale and Baltimore, Cell, 57: 103-113 (1989)]. There aretwo such consensus sequences in the MN putative promoter; however, theydo not overlap the transcription start (FIG. 6).

An interesting region was found in the middle of the MN gene. The regionis about 1.4 kb in length [nt 4,600-6,000 of the genomic sequence; SEQID NO: 49] and spans from the 3′ part of the 1st intron to the end ofthe 5th exon. The region has the character of a typical CpG-rich island,with 62.8% C+G content and 82 CpG: 131 GpC dinucleotides. Moreover,there are multiple putative binding sites for transcription factors AP2and Sp1 [Locker and Buzard, supra; Briggs et al., Science, 234: 47-52(1986)] concentrated in the center of this area. Particularly the 3rdintron of 131 bp in length contains three Sp1 and three AP2 consensussequences. That data indicates the possible involvement of that regionin the regulation of MN gene expression. However, functionality of thatregion, as well as other regulatory elements found in the proposed 5′ MNpromoter, remains to be determined.

MN Promoter

Study of the MN promoter has shown that it is TATA-less and containsregulatory sequences for AP-1, AP-2, as well as two p53 binding sites.The sequence of the 5′ end of the 3.5 kb flanking region upstream of theMN gene has shown extensive homology to LTR of HERV-K endogenousretroviruses. Basal transcription activity of the promoter is very weakas proven by analyses using CAT and neo reporter genes. However,expression of the reporter genes is several fold increased when drivenfrom the 3.5 kb flanking region, indicating involvement of putativeenhancers.

Functional characterization of the 3.5 kb MN 5′ upstream region bydeletion analysis lead to the identification of the [−173, +31] fragment[SEQ ID NO: 21] (also alternatively, but less preferably, the nearlyidentical −172, +31 fragment [SEQ ID NO: 91]) as the MN promoter. Invitro DNase I footprinting revealed the presence of five protectedregions (PR) within the MN promoter. Detailed deletion analysis of thepromoter identified PR 1 and 2 (numbered from the transcription start)as the most critical for transcriptional activity. PR4 [SEQ ID NO: 115]negatively affected transcription as its deletion led to increasedpromoter activity and was confirmed to function as a promoter-,position- and orientation-independent silencer element. Mutationalanalysis indicated that the direct repeat AGGGCacAGGGC [SEQ ID NO: 143]is required for efficient repressor binding. Two components of therepressor complex (35 and 42 kDa) were found to be in direct contactwith PR4 by UV crosslinking. Increased cell density, known to induce MNexpression, did not affect levels of PR4 binding in HeLa cells.Significantly reduced repressor level seems to be responsible for MNup-regulation in the case of tumorigenic CGL3 as compared tonon-tumorigenic CGL1 HeLa×normal fibroblast hybrid cells.

Utility of MN Promoter and MN Promoter Fragments as Tumor-SpecificPromoters for Gene Therapy

Being investigated is whether the MN gene promoter and MN/CA 9 promoterfragments can be used as tumor-specific promoters to drive theexpression of a suicide gene [for example, thymidine kinase (tk) ofHSV)] and mediate the direct and bystander killing of tumor cells. HSVtkgene transferred to tumor cells converts nucleoside analogue ganciclovir(GCV) to toxic triphosphates and mediates the death of transduced andalso neighboring tumor cells. The control of HSVtk by the MN genepromoter or a MN/CA 9 promoter fragment would allow its expression onlyin tumor cells, which are permissive for the biosynthesis of MN protein,and selectively kill such tumor cells, but not normal cells in which MNexpression is repressed.

A plasmid construct in which HSVtk was cloned downstream of the MNpromoter region Bd3, containing both proximal and distant regulatoryelements of MN, was prepared. That plasmid pMN-HSVtk was transfected toRat2TK− cells and C33 human cervical carcinoma cells using calciumphosphate precipitation and lipofection, respectively. Transfectantswere tested for expression of HSVtk and GVC sensitivity. Analysis of thetransfectants has shown the remarkable cytotoxic in vitro effect of GVCeven in low concentrations (up to 95% of cells killed).

Polyclonal rabbit antiserum against HSVtk, using fusion protein with GSTin pGEX-3X, has been prepared to immunodetect HSVtk synthesized intransfected cells. This model system is being studied to estimate thebystander effect, the inhibition of cloning efficiency and invasivenessof transduced and GVC-treated cells to collagen matrices. A recombinantretroviral vector with the MN promoter-driven HSVtk is to be prepared totest its in vivo efficacy using an animal model (e.g., SCID-mouse).

MN/CA 9 and Hypoxia

Particularly relied upon herein in regard to aspects of this inventionthat relate to MN/CA 9 and hypoxia, and MN/CA 9's HRE are the followingarticles incorporated in U.S. Provisional Application 60/341036 (filedDec. 13, 2001), from which the instant application claims priority:

-   Wykoff et al., “Hypoxia-inducible Expression of Tumor-associated    Carbonic Anhydrases,” Cancer Research, 60(24): 7075-7083 (Dec. 15,    2000)-   Turner et al., “The hypoxia induced genes VEGF (vascular endothelial    growth factor) and CA9 (carbonic anhydrase 9) are differentially    regulated in superficial vs invasive human bladder cancer,” European    Urology, 39(Supp. 5): pp. 171 (March 2001)-   Wykoff et al., “Expression of the Hypoxia-Inducible and    Tumor-Associated Carbonic Anhydrases in Ductal Carcinoma in Situ of    the Breast,” American Journal of Pathology, 158(3): 1011-1019 (March    2001)-   Beasley et al., “Carbonic Anhydrase IX, an Endogenous Hypoxia    Marker, Expression in Head and Neck Squamous Cell Carcinoma and its    Relationship to Hypoxia, Necrosis, and Microvessel Density,” Cancer    Research, 61(13): 5262-5267 (Jul. 1, 2001)-   Harris, A. L., “Hypoxia regulated transcriptome: Implications for    tumour angiogenesis and therapy,” British Journal of Cancer,    85(Supp. 1): pp. 4 (July 2001)-   Chia et al., “Prognostic Significance of a Novel Hypoxia-Regulated    Marker, Carbonic Anhydrase IX, in Invasive Breast Carcinoma,”    Journal of Clinical Oncology, 19(16): 3660-3668 (Aug. 15, 2001)-   Loncaster et al., “Carbonic Anhydrase (CA IX) Expression, a    Potential New Intrinsic Marker of Hypoxia: Correlations with Tumor    Oxygen Measurements and Prognosis in Locally Advanced Carcinoma of    the Cervix,” Cancer Research, 61(17): 6394-6399 (Sep. 1, 2001)-   Giatromanolaki et al., “Expression of Hypoxia-inducible Carbonic    Anhydrase-9 Relates to Angiogenic Pathways and Independently to Poor    Outcome in Non-Small Cell Lung Cancer,” Cancer Research, 61(21):    7992-7998 (Nov. 1, 2001)-   Koukourakis et al., “Hypoxia-regulated Carbonic Anhydrase-9 (CA9)    Relates to Poor Vascularization and Resistance of Squamous Cell Head    and Neck Cancer to Chemoradiotherapy,” Clinical Cancer Research,    7(11): 3399-3403 (November 2001)-   O'Byrne et al., “Towards a biological staging model for operable    non-small cell lung cancer,” Lung Cancer, 34(Supp. 2): S83-S89    (December 2001)    The above-listed articles and the references cited therein are    hereby incorporated by reference

Studies of the MN/CA 9 promoter demonstrated that the hypoxia-inducibleresponse is mediated by HIF, and that it is dependent on a consensus HREor consensus HBS (depending upon the terminology applied) lying adjacentto the initiation site. Studies of the MN/CA 9 promoter alsodemonstrated that promoter fragments close to the transcriptioninitiation site were sufficient to convey a hypoxia-inducible response.

The MN/CA 9 promoter contains neither a TATA box nor a consensusinitiator sequence at the cap site. The association of that unusualanatomy with tight regulation by hypoxia renders MN/CA 9 of particularclinical interest. Also unusual and of particular clinical interest isthe strong hypoxia-inducibility conveyed by the minimal MN/CA 9 promoter(−36/+14) [SEQ ID NO: 146] and its putative HRE [SEQ ID NO: 145] or HBS[alternatively, SEQ ID NOS: 145, 147, 148, 149 or 150, most preferablySEQ ID NO: 145]. The MN/CA 9 promoter HRE or HBS comprised in variousMN/CA 9 promoter fragments may be of considerable utility in therefinement of gene therapy vectors seeking to target therapeutic geneexpression to hypoxic regions of tumors. (Wykoff et al. 2000).

Further refinement can be envisioned by one of skill in the art bypositioning enhancer elements strategically within a MN/CA 9 promoterfragment comprising a HRE/HBS. Still further refinement could beenvisioned as placing the MN/CA 9 HRE/HBS and associated flankingsequences within a promoter for another gene as considered to bestrategically advantageous.

For example, Dachs et al. Nat. Med., 3: 515-520 (1997) describes invitro experiments in which a PGK-1 HRE promoter is used to drive theexpression of a bacterial cytosine deaminase gene, which gene product inturn activates the prodrug 5-fluorocytosine (5-FC) to 5-fluorouracil(5-FU). The overall effect is to sensitize human cells to the prodrug,to which they are normally resistant. A similar system could be appliedto hypoxic cells, in order to selectively sensitize tumor hypoxic cellsto a prodrug by transfecting them with an activating gene driven by anMN/CA 9 HRE and promoter, followed by treatment with the prodrug. Theadvantage of using the MN/CA 9 HRE and promoter, as opposed to otherHIF-regulated genes, is that MN/CA 9 expression correlates uniquely wellwith both tumor necrosis and low pO₂ tension.

Because of the unusually tight regulation of the MN/CA9 gene by hypoxia,the hypoxia-response element of the MN/CA 9 gene is considered to beuseful to determine HIF activation, either by microenvironmental hypoxiaor genetic events such as VHL inactivation.

Although other hypoxia-induced proteins may be useful markers ofhypoxia, MN/CA IX is induced at the same oxygen tension at which HIF-1αis induced and provides a measure of the percentage of the tumorpopulation that is hypoxic (Beasley et al., 2001). MN/CA 9 expressioncorrelates with the oxygen diffusion distance and is expressed in aperinecrotic manner in head and neck squamous cell carcinoma (HNSCC).

To investigate the unusually tight regulation of MN/CA 9 mRNA byhypoxia, the oxygen-dependent function of the MN/CA 9 promoter wastested. Mutational analysis of the MN/CA 9 hypoxia-response elementsequence was performed in HeLa and CHO cell lines. Transienttransfection experiments were performed using reporter plasmidscontaining full or partial sequences lying about 0.1 kb 5′ to theluciferase reporter gene. Mutations were made within the consensus HRE(or HBS) sequence to confirm the importance of the putative MN/CA 9 HRE.

The invention provides in one aspect for the MN/CA 9 HRE sequence to beused in a vector as described herein in the treatment of a patient witha hypoxia-related condition. Such vectors according to the invention canbe administered by injection of the vector construct directly into asolid tumor, in the form of naked nucleic acid, preferably DNA, vectors.Alternatively, other vectors such as retroviruses may be used. Accordingto the invention, the vector containing the MN/CA 9 HRE sequence may beinjected into the solid tumor, followed by administration of a prodrugin the case of a vector encoding a pro-drug activation system.

In one embodiment of the invention, the vector containing the MN/CA 9HRE sequence may be used in treatment of solid tumors. Alternatively,the vector containing the MN/CA 9 HRE sequence may be used in treatmentof other types of diseases where target cells are affected by hypoxia,such as acute and chronic vascular disease and pulmonary disease. Forexample, the gene regulated by the MN/CA 9 HRE may encode for a cytokineor a growth factor. A vascular growth factor can be used to stimulateangiogenesis in hypoxic areas.

In another embodiment of the invention, the vector containing the MN/CA9 HRE sequence may be used to monitor or measure levels of hypoxia.Examples 6-9 below further elucidate the relationship between MN/CA 9and hypoxia, and aspects of this invention relating thereto.

MN Promoter Analysis

Since the MN promoter is weak, a classical approach to study it would belimited due to the relatively low efficiency of transient transfections(up to 10%). Therefore, stable clonal cell lines expressing constructscontaining the MN promoter fused to the CAT gene were prepared. In suchclonal lines, 100% of the cells express the CAT gene driven from the MNpromoter, and thus, the activity of the promoter is detectable easierthan in transient experiments. Also, the promoter activity can beanalysed repeatedly in the same cells under different conditions ortreated by different factors and drugs. This approach allows for thestudy of the mechanisms underlying MN regulation at the level oftranscription initiation.

Several types of transfections were performed with promoter constructslinked to a reporter CAT gene (calcium precipitation, DEAE dextrancombined with DMSO shock and/or chloroquine, as well aselectroporation), using different methods of CAT activity assay(scintillation method, thin layer chromatography) and several recipientcell lines differing in the level of MN expression and in transfectionefficiency (HeLa, SiHa, CGL3, KATO III, Rat2TK⁻ and C33 cells). Activityof the MN promoter was detected preferably by the electroporation ofCGL3 cells and thin layer chromatography. Further preferably, C33 cellscotransfected with MN promoter-CAT constructs and pSV2neo were used.

1. To detect basal activity of the MN promoter and to estimate theposition of the core promoter, expression of the CAT gene fromconstructs pMN1 to pMN7 after transfection to CGL3 cells was analyzed.Plasmids with progressive 5′ deletions were transfected into CGL3 cellsand activity was analyzed by CAT assay. [8 μg of DNA was used fortransfection in all cases except pBLV-LTR (2 μg).]

Only very weak CAT activity was detected in cells transfected by pMN1and pMN2 (containing respectively 933 bp and 600 bp of the promotersequence). A little higher activity was exhibited with the constructspMN3, pMN4 and pMN6 (containing respectively 446 bp, 243 bp and 58 bp ofthe promoter). A slight peak of activity was obtained with pMN5(starting at position −172 with respect to the transcription start.)Thus, the function of the MN core promoter can be assigned to a regionof approximately 500 bp immediately upstream from the MN transcriptioninitiation site.

Interestingly, the activity of the large Bd3 region (covering 3.5 kbpupstream of the transcription start) was severalfold higher than theactivity of the core promoter. However, its level was still much lowerthan that exhibited by a positive control, i.e., BLV-LTR transactivatedby Tax, and even lower than the activity of BLV-LTR withouttransactivation. That the activity of Bd3 was elevated in comparison tothe core promoter suggests the presence of some regulatory elements.Such elements are most probably situated in the sequence between pMN1and Bd3 (i.e. from −1 kbp to −3.5 kbp) [SEQ ID NO: 58]. The cloning andtransfection of several deletion versions of Bd3 covering the indicatedregion can be used to determine the location of the putative regulatoryelements.

Similar results were obtained from transfecting KATO III cells with Bd3and pMN4. The transfected cells expressed a lower level of MN than theCGL3 cells. Accordingly, the activity of the MN promoter was found to belower than in CGL3 cells.

2. In a parallel approach to study the MN promoter, an analysis based onG418 selection of cells transfected by plasmids containing the promoterof interest cloned upstream from the neo gene was made. This approach issuitable to study weak promoters, since its sensitivity is much higherthan that of a standard CAT assay. The principle underlying the methodis as follows: an active promoter drives expression of the neo genewhich protects transfected cells from the toxic effect of G418, whereasan inactive promoter results in no neo product being made and the cellstransfected thereby die upon the action of G418. Therefore, the activityof the promoter can be estimated according to the number of cellcolonies obtained after two weeks of selection with G418. Threeconstructs were used in the initial experiments—pMN1neo, pMN4neo andpMN7neo. As pMN7neo contains only 30 bp upstream of the transcriptionstart site, it was considered a negative control. As a positive control,pSV2neo with a promoter derived from SV40 was used. Rat2TK⁻ cells werechosen as the recipient cells, since they are transfectable with highefficiency by the calcium precipitation method.

After transfection, the cells were subjected to two weeks of selection.Then the medium was removed, the cells were rinsed with PBS, and thecolonies were rendered visible by staining with methylene blue. Theresults obtained from three independent experiments corroborated thedata from the CAT assays. The promoter construct pMN4neo exhibitedhigher transcriptional activity than pMN1 neo. However, the differencebetween the positive control and pMN4neo was not so striking as in theCAT assay. That may have been due to both lower promoter activity ofpSV2neo compared to Tax-transactivated pBLV-LTR and to differentconditions for cell growth after transfection. From that point of view,stable transfection is probably more advantageous for MN expression,since the cells grow in colonies with close cell to cell contact, andthe experiment lasts much longer, providing a better opportunity todetect promoter activity.

3. Stable transfectants expressing MN promoter-CAT chimeric genes wereprepared by the cotransfection of relevant plasmids with pSV2neo. Asrecipient cells, HeLa cells were used first. However, no clonesexpressing the promoter-CAT constructs were obtained. That negativeresult was probably caused by homologic recombination of the transfectedgenomic region of MN (e.g. the promoter) with the correspondingendogenous sequence. On the basis of that experience, C33 cells derivedfrom a HPV-negative cervical carcinoma were used. C33 cells do notexpress MN, since during the process of tumorigenesis, they lost geneticmaterial including chromosomal region 9p which contains the MN gene. Inthese experiments, the absence of the MN gene may represent an advantageas the possibility of homologic recombinations is avoided.

C33 Cells Transfected with MN Promoter-CAT Constructs

C33 cells expressing the CAT gene under MN promoter regions Bd3(−3500/+31) [SEQ ID NO: 90] and MN5 (−172/+31) [SEQ ID NO: 91] were usedfor initial experiments to analyze the influence of cell density on thetranscriptional activity of the MN promoter. The results indicated thatsignals generated after cells come into close contact activatetranscription of the CAT protein from the MN promoter in proportion tothe density of the cell culture. Interestingly, the data indicated thatthe MN protein is not required for this phase of signal transduction,since the influence of density is clearly demonstrated in MN-negativeC33 cells. Rather, it appears that MN protein acts as an effectormolecule produced in dense cells in order to perform a certainbiological function (i.e., to perturb contact inhibition). Alsointerestingly, the MN promoter activity is detectable even in verysparse cell cultures suggesting that MN is expressed at a very low levelalso is sparse subconfluent culture.

Deletion Variants. Deletion variants of the Bd3-CAT promoter constructwere then prepared. The constructs were cotransfected with pSV2neo intoC33 cervical cells. After selection with G418, the whole population ofstably transfected cells were subjected to CAT ELISA analysis.Expression of the deletion constructs resulted in the synthesis ofsimilar levels of CAT protein to that obtained with the Bd3-CATconstruct. On the basis of that preliminary data, the inventors proposedthat sequences stimulating transcription of MN are located between −3506and −3375 bp [SEQ ID NO: 92] upstream from the transcription start. Thatis the sequence exhibiting homology to HERV-K LTR.

However, transient transfection studies in CGL3 cells repeatedlyrevealed that the LTR region is not required for the enhancement ofbasal MN promoter activity. Further, results obtained in CGL3 cellsindicate that the activating element is localized in the region from−933 to −2179 [SEQ ID NO: 110] with respect to transcription initiationsite (the position of the region having been deduced from overlappingsequences in the Bd3 deletion mutants).

Interaction of Nuclear Proteins with MN Promoter Sequences

In order to identify transcription factors binding to the MN promoterand potentially regulating its activity, a series of analyses using anelectrophoretic mobility shift assay (EMSA) and DNase I footprintinganalysis (FTP) were performed.

EMSA

In the EMSA, purified promoter fragments MN4 (−243/+31) [SEQ ID NO: 93],MN5 (−172/+31) [SEQ ID NO: 91], MN6 (−58/+31) [SEQ ID NO: 94] and MN7(−30/+31) [SEQ ID NO: 95], labeled at the 3′ ends by Klenow enzyme, wereallowed to interact with proteins in nuclear extracts prepared from CGL1and CGL3 cells. [40 μg of nuclear proteins were incubated with 30,000cpm end-labeled DNA fragments in the presence of 2 μg poly(dIdC).]DNA-protein complexes were analysed by PAGE (native 6%), where thecomplexes created extra bands that migrated more slowly than the freeDNA fragments, due to the shift in mobility which is dependent on themoiety of bound protein.

The EMSA of the MN4 and MN5 promoter fragments revealed severalDNA-protein complexes; however, the binding patterns obtainedrespectively with CGL1 and CGL3 nuclear extracts were not identical.There is a single CGL-1 specific complex.

The EMSA of the MN6 promoter fragment resulted in the formation of threeidentical complexes with both CGL1 and CGL3 nuclear extracts, whereasthe MN7 promoter fragment did not bind any nuclear proteins.

The EMSA results indicated that the CGL1 nuclear extract contains aspecific factor, which could participate in the negative regulation ofMN expression in CGL1 cells. Since the specific DNA-protein complex isformed with MN4 (−243/+31) [SEQ. ID NO.: 93] and MN5 (−172/+31) [SEQ. IDNO.: 91] promoter fragments, but not with MN6 (−58/+31) [SEQ ID NO: 94],it appears that the binding site of the protein component of thatspecific complex is located between −173 and −58 bp [SEQ. ID NO.: 96]with respect to transcription initiation.

The next step was a series of EMSA analyses using double stranded (ds)oligonucleotides designed according to the protected regions in FTPanalysis. A ds oligonucleotide derived from the protected region PR2[covering the sequence from −72 to −56 bp (SEQ ID NO: 111)] of the MNpromoter provided confirmation of the binding of the AP-1 transcriptionfactor in competitive EMSA using commercial ds olignucleotidesrepresenting the binding site for AP-1.

EMSA of ds oligonucleotides derived from the protected regions of PR1[−46 to −24 bp (SEQ ID NO: 112)], PR2 [−72 to −56 bp (SEQ ID NO: 111)],PR3 [−102 to −85 (SEQ ID NO: 113)] and PR5 [−163 to −144 (SEQ ID NO:114)] did not reveal any differences in the binding pattern of nuclearproteins extracted from CGL1 and CGL3 cells, indicating that thoseregions do not bind crucial transcription factors which controlactivation of the MN gene in CGL3, or its negative regulation in CGL1.However, EMSA of ds oligonucleotides from the protected region PR4 [−133to −108; SEQ ID NO: 115] repeatedly showed remarkable quantitativedifferences between binding of CGL1 and CGL3 nuclear proteins. CGL1nuclear proteins formed a substantially higher amount of DNA-proteincomplexes, indicating that the PR4 region contains a binding site forspecific transcription factor(s) that may represent a negative regulatorof MN gene transcription in CGL1 cells. That fact is in accord with theprevious EMSA data which showed CGL-1 specific DNA-protein complex withthe promoter fragments pMN4 (−243/+31; SEQ ID NO: 93) and pMN5(−172/+31; SEQ ID NO: 91), but not with pMN6 (−58/+31; SEQ ID NO: 94).

To identify the protein involved or the formation of a specific complexwith the MN promoter in the PR4 region, relevant ds oligonucleotidescovalently bound to magnetic beads will be used to purify thecorresponding transcription factor. Alternatively the ONE Hybrid System®[Clontech (Palo Alto, Calif. (USA)] will be used to search for and clonetranscription factors involved in regulation of the analysed promoterregion. A cDNA library from HeLa cells will be used for thatinvestigation.

FTP

To determine the precise location of cis regulatory elements thatparticipate in the transcriptional regulation of the MN gene, FTP wasused. Proteins in nuclear extracts prepared respectively from CGL1 andCGL3 cells were allowed to interact with a purified ds DNA fragment ofthe MN promoter (MN4, −243/+31) [SEQ ID NO: 93] which was labeled at the5′ end of one strand. [MN4 fragments were labeled either at Xho1 site(−243/+31*) or at Xba1 site (*−243/+31).] The DNA-protein complex wasthen subjected to DNase I attack, which causes the DNA chain to break atcertain bases if they are not in contact with proteins. [A control usedBSA instead of DNase.] Examination of the band pattern of the denaturedDNA after gel electrophoresis [8% denaturing gel] indicates which of thebases on the labeled strand were protected by protein.

FTP analysis of the MN4 promoter fragment revealed 5 regions (I-V)protected at both the coding and noncoding strand, as well as tworegions (VI and VII) protected at the coding strand but not at thenoncoding strand. FIG. 6 indicates the general regions on the MNpromoter that were protected.

The sequences of the identified protected regions (PR) were subjected tocomputer analysis using the SIGNALSCAN program to see if theycorresponded to known consensus sequences for transcription factors. Thedata obtained by that computer analyses are as follows:

PR I coding strand - AP-2, p53, GAL4 noncoding strand - JCV-repeated PRII coding strand - AP-1, CGN4 noncoding strand - TCF-1, dFRA, CGN4 PRIII coding strand - no known consensus sequence, only partial overlap ofAP1 noncoding strand - 2 TCF-1 sites PR IV coding strand - TCF-1, ADR-1noncoding strand - CTCF, LF-A1, LBP-1 PR V coding strand - no knownconsensus motif noncoding strand - JCV repeated PR VI coding strand - noknown consensus motif noncoding strand - T antigen of SV 40, GAL4 PR VIIcoding strand - NF-uE4, U2snRNA.2 noncoding strand - AP-2, IgHC.12,MyoD.

In contrast to EMSA, the FTP analysis did not find any differencesbetween CGL1 and CGL3 nuclear extracts. However, the presence ofspecific DNA-protein interactions detected in the CGL1 nuclear extractsby EMSA could have resulted from the binding of additional protein toform DNA protein-protein complex. If that specific protein did notcontact the DNA sequence directly, its presence would not be detectableby FTP.

EMSA Supershift Analysis

The results of the FTP suggests that transcription factors AP-1, AP-2 aswell as tumor suppressor protein p53 are potentially involved in theregulation of MN expression. To confirm binding of those particularproteins to the MN promoter, a supershift analysis using antibodiesspecific for those proteins was performed. For this analysis,DNA-protein complexes prepared as described for EMSA were allowed tointeract with MAbs or polyclonal antibodies specific for proteinspotentially included in the complex. The binding of antibody to thecorresponding protein results in an additional shift (supershift) inmobility of the DNA-protein-antibody complex which is PAGE visualized asan additional, more slowly migrating band.

By this method, the binding of AP-2 to the MN promoter was confirmed.However, this method did not evidence binding of the AP-1 transcriptionfactor. It is possible that MN protein binds AP-1-related protein, whichis antigenically different from the AP-1 recognized by the antibodiesused in this assay.

Also of high interest is the possible binding of the p53 tumorsuppressor protein to the MN promoter. It is well known that wt p53functions as a transcription factor, which activates expression ofgrowth-restricting genes and down-modulates, directly or indirectly, theexpression of genes that are required for ongoing cell proliferation.Transient co-transfection experiments using the pMN4-CAT promoterconstruct in combination with wt p53 cDNA and mut p53 cDNA,respectively, suggested that wt p53, but not mut p53, negativelyregulates expression of MN. In addition, one of two p53-binding sites inthe MN promoter is protected in FTP analysis (FIG. 6), indicating thatit binds to the corresponding protein. Therefore, supershift analysis toprove that p53 binds to the MN promoter with two p53-specificantibodies, e.g. Mabs 421 and DO-1 [the latter kindly provided by Dr.Vojtesek from Masaryk Memorial Cancer Institute in Brno, Czech Republic]are to be performed with appropriate nuclear extracts, e.g. from MCF-7breast carcinoma cells which express wt p53 at a sufficient level.

Regulation of MN Expression and MN Promoter

MN appears to be a novel regulatory protein that is directly involved inthe control of cell proliferation and in cellular transformation. InHeLa cells, the expression of MN is positively regulated by celldensity. Its level is increased by persistent infection with LCMV. Inhybrid cells between HeLa and normal fibroblasts, MN expressioncorrelates with tumorigenicity. The fact that MN is not present innontumorigenic hybrid cells (CGL1), but is expressed in a tumorigenicsegregant lacking chromosome 11, indicates that MN is negativelyregulated by a putative suppressor in chromosome 11.

Evidence supporting the regulatory role of MN protein was found in thegeneration of stable transfectants of NIH 3T3 cells that constitutivelyexpress MN protein. As a consequence of MN expression, the NIH 3T3 cellsacquired features associated with a transformed phenotype: alteredmorphology, increased saturation density, proliferative advantage inserum-reduced media, enhanced DNA synthesis and capacity foranchorage-independent growth. Further, flow cytometric analyses ofasynchronous cell populations indicated that the expression of MNprotein leads to accelerated progression of cells through G1 phase,reduction of cell size and the loss of capacity for growth arrest underinappropriate conditions. Also, MN expressing cells display a decreasedsensitivity to the DNA damaging drug mitomycin C.

Nontumorigenic human cells, CGL1 cells, were also transfected with thefull-length MN cDNA. The same pSG5C-MN construct in combination withpSV2neo plasmid as used to transfect the NIH 3T3 cells was used. Out of15 MN-positive clones (tested by SP-RIA and Western blotting), 3 werechosen for further analysis. Two MN-negative clones isolated from CGL1cells transfected with empty plasmid were added as controls. Initialanalysis indicates that the morphology and growth habits ofMN-transfected CGL1 cells are not changed dramatically, but theirproliferation rate and plating efficiency is increased.

MN Promoter—Sense/Antisense Constructs

When the promoter region from the MN genomic clone, isolated asdescribed above, was linked to MN cDNA and transfected into CGL1 hybridcells, expression of MN protein was detectable immediately afterselection. However, then it gradually ceased, indicating thus an actionof a feedback regulator. The putative regulatory element appeared to beacting via the MN promoter, because when the full-length cDNA (notcontaining the promoter) was used for transfection, no similar effectwas observed.

An “antisense” MN cDNA/MN promoter construct was used to transfect CGL3cells. The effect was the opposite of that of the CGL1 cells transfectedwith the “sense” construct. Whereas the transfected CGL1 cells formedcolonies several times larger than the control CGL1, the transfectedCGL3 cells formed colonies much smaller than the control CGL3 cells. Thesame result was obtained by antisense MN cDNA transfection in SiHa andHeLa cells.

For those experiments, the part of the promoter region that was linkedto the MN cDNA through a BamHI site was derived from a NcoI-BamHIfragment of the MN genomic clone [Bd3] and represents a region a fewhundred bp upstream from the transcription initiation site. After theligation, the joint DNA was inserted into a pBK-CMV expression vector[Stratagene]. The required orientation of the inserted sequence wasensured by directional cloning and subsequently verified by restrictionanalysis. The tranfection procedure was the same as used in transfectingthe NIH 3T3 cells, but co-transfection with the pSV2neo plasmid was notnecessary since the neo selection marker was already included in thepBK-CMV vector.

After two weeks of selection in a medium containing G418, remarkabledifferences between the numbers and sizes of the colonies grown wereevident as noted above. Immediately following the selection and cloning,the MN-transfected CGL1 and CGL3 cells were tested by SP-RIA forexpression and repression of MN, respectively. The isolated transfectedCGL1 clones were MN positive (although the level was lower than obtainedwith the full-length cDNA), whereas MN protein was almost absent fromthe transfected CGL3 clones. However, in subsequent passages, theexpression of MN in transfected CGL1 cells started to cease, and wasthen blocked perhaps evidencing a control feedback mechanism.

As a result of the very much lowered proliferation of the transfectedCGL3 cells, it was difficult to expand the majority of cloned cells(according to SP-RIA, those with the lowest levels of MN), and they werelost during passaging. However, some clones overcame that problem andagain expressed MN. It is possible that once those cells reached ahigher quantity, that the level of endogenously produced MN mRNAincreased over the amount of ectopically expressed antisense mRNA.

Identification of Specific Transcription Factors Involved in Control ofMN Expression

Control of MN expression at the transcription level involves regulatoryelements of the MN promoter. Those elements bind transcription factorsthat are responsible for MN activation in tumor cells and/or repressionin normal cells. The identification and isolation of those specifictranscription factors and an understanding of how they regulate MNexpression could result in their therapeutic utility in modulating MNexpression.

EMSA experiments indicate the existence of an MN gene repressor. We usedthe One Hybrid System® [Clontech (Palo Alto, Calif.), an in vivo yeastgenetic assay for isolating genes encoding proteins that bind to atarget, cis-acting regulatory element or any other short DNA-bindingsequence; (Fields and Song, Nature, 340: 245 (1989): Wu et al., EMBO J.,13: 4823 (1994)] and subtractive suppressive PCR (SSH). SSH allows thecloning of genes that are differentially expressed under conditionswhich are known to up or down regulate MN expression such as densityversus sparsity of HeLa cells, and suspension versus adherent HeLacells.

In experiments with HPV immobilized cervical cells (HCE 16/3), it wasfound that the regulation of MN expression differs from that in fullytransformed carcinoma cells. For example, glucocorticoid hormones, whichactivate HPV transcription, negatively regulate MN expression in HCE,but stimulate MN in HeLa and SiHa. Further keratinocyte growth factors,which down regulates transcription of HPV oncogenes, stimulates MNexpression in suspension HCE but not in adherent cells.

EGF and insulin are involved in the activation of MN expression in bothimmortalized and carcinoma cells. All the noted facts can be used in thesearch for MN-specific transcription factors and in the modulation of MNexpression for therapeutic purposes.

Deduced Amino Acid Sequence

The ORF of the MN cDNA shown in FIG. 1 has the coding capacity for a 459amino acid protein with a calculated molecular weight of 49.7 kd. Theoverall amino acid composition of the MN/CA IX protein is rather acidic,and predicted to have a pl of 4.3. Analysis of native MN/CA IX proteinfrom CGL3 cells by two-dimensional electrophoresis followed byimmunoblotting has shown that in agreement with computer prediction, theMN/CA IX is an acidic protein existing in several isoelectric forms withpls ranging from 4.7 to 6.3.

As assessed by amino acid sequence analysis, the deduced primarystructure of the MN protein can be divided into four distinct regions.The initial hydrophobic region of 37 amino acids (aa) corresponds to asignal peptide. The mature protein has an N-terminal or extracellularpart of 377 amino acids [aa 38-414 (SEQ ID NO: 87], a hydrophobictransmembrane segment of 20 amino acids [aa 415-434 (SEQ ID NO: 52)] anda C-terminal region of 25 amino acids [aa 435-459 (SEQ ID NO: 53)].

The extracellular part is composed of two distinct domains: (1) aproteoglycan-like domain [aa 53-111 (SEQ ID NO: 50)]; and (2) a CAdomain, located close to the plasma membrane [aa 135-391 (SEQ ID NO:51)]. [The amino acid numbers are keyed to those of FIG. 1.]

More detailed insight into MN protein primary structure disclosed thepresence of several consensus sequences. One potential N-glycosylationsite was found at position 346 of FIG. 1. That feature, together with apredicted membrane-spanning region are consistent with the results, inwhich MN was shown to be an N-glycosylated protein localized in theplasma membrane. MN protein sequence deduced from cDNA was also found tocontain seven S/TPXX sequence elements [SEQ ID NOS: 25 AND 26] (one ofthem is in the signal peptide) defined by Suzuki, J. Mol. Biol., 207:61-84 (1989) as motifs frequently found in gene regulatory proteins.However, only two of them are composed of the suggested consensus aminoacids.

Experiments have shown that the MN protein is able to bind zinc cations,as shown by affinity chromatography using Zn-charged chelatingsepharose. MN protein immunoprecipitated from HeLa cells by Mab M75 wasfound to have weak catalytic activity of CA. The CA-like domain of MNhas a structural predisposition to serve as a binding site for smallsoluble domains. Thus, MN protein could mediate some kind of signaltransduction.

MN protein from LCMV-infected HeLA cells was shown by using DNAcellulose affinity chromatography to bind to immobilized double-strandedsalmon sperm DNA. The binding activity required both the presence ofzinc cations and the absence of a reducing agent in the binding buffer.

CA Domain Required for Anchorage Independence but for IncreasedProliferation of Transfected NIH 3T3 Fibroblasts

In transfected NIH 3T3 fibroblasts, MN protein induces morphologictransformation, increased proliferation and anchorage independence. Theconsequences of constitutive expression of two MN-truncated variants inNIH 3T3 cells were studied. It was found that the proteoglycan-likeregion is sufficient for the morphological alteration of transfectedcells and displays the growth-promoting activity presumably related toperturbation of contact inhibition.

The CA domain is essential for induction of anchorage independence,whereas the TM anchor and IC tail are dispensable for that biologicaleffect. The MN protein is also capable of causing plasma membraneruffling in the transfected cells and appears to participate in theirattachment to the solid support. The data evince the involvement of MNin the regulation of cell proliferation, adhesion and intercellularcommunication.

Sequence Similarities

Computer analysis of the MN cDNA sequence was carried out using DNASISand PROSIS (Pharmacia Software packages). GenBank, EMBL, ProteinIdentification Resource and SWISS-PROT databases were searched for allpossible sequence similarities. In addition, a search for proteinssharing sequence similarities with MN was performed in the MIPS databankwith the FastA program [Pearson and Lipman, PNAS (USA), 85: 2444(1988)].

The proteoglycan-like domain [aa 53-111 (SEQ ID NO: 50)], which isbetween the signal peptide and the CA domain, shows significant homology(38% identity and 44% positivity) with a keratan sulphate attachmentdomain of a human large aggregating proteoglycan aggrecan [Doege et al.,J. Biol. Chem., 266: 894-902 (1991)].

The CA domain [aa 135-391 (SEQ ID NO: 51)] is spread over 265 aa andshows 38.9% amino acid identity with the human CA VI isoenzyme [Aldredet al., Biochemistry, 30: 569-575 (1991)]. The homology between MN/CA IXand other isoenzymes is as follows: 35.2% with CA II in a 261 aa overlap[Montgomery et al., Nucl. Acids. Res., 15: 4687 (1987)], 31.8% with CA Iin a 261 aa overlap [Barlow et al., Nucl. Acids Res., 15: 2386 (1987)],31.6% with CA IV in a 266 aa overlap [Okuyama et al., PNAS (USA) 89:1315-1319 (1992)], and 30.5% with CA III in a 259 aa overlap (Lloyd etal., Genes. Dev., 1: 594-602 (1987)].

In addition to the CA domain, MN/CA IX has acquired both N-terminal andC-terminal extensions that are unrelated to the other CA isoenzymes. Theamino acid sequence of the C-terminal part, consisting of thetransmembrane anchor and the intracytoplasmic tail, shows no significanthomology to any known protein sequence.

The MN gene was clearly found to be a novel sequence derived from thehuman genome. The overall sequence homology between the cDNA MN sequenceand cDNA sequences encoding different CA isoenzymes is in a homologyrange of 48-50% which is considered by ones in the art to be low.Therefore, the MN cDNA sequence is not closely related to any CA cDNAsequences.

Only very closely related nt sequences having a homology of at least80-90% would hybridize to each other under stringent conditions. Asequence comparison of the MN cDNA sequence shown in FIG. 1 and acorresponding cDNA of the human carbonic anhydrase II (CA II) showedthat there are no stretches of identity between the two sequences thatwould be long enough to allow for a segment of the CA II cDNA sequencehaving 25 or more nucleotides to hybridize under stringent hybridizationconditions to the MN cDNA or vice versa.

A search for nt sequences related to MN gene in the EMBL Data Librarydid not reveal any specific homology except for 6 complete and 2 partialAlu-type repeats with homology to Alu sequences ranging from 69.8% to91% [Jurka and Milosavljevic, J. Mol. Evol. 32: 105-121 (1991)]. Also a222 bp sequence proximal to the 5′ end of the genomic region is shown tobe closely homologous to a region of the HERV-K LTR.

In general, nucleotide sequences that are not in the Alu or LTR-likeregions, of preferably 25 bases or more, or still more preferably of 50bases or more, can be routinely tested and screened and found tohybridize under stringent conditions to only MN nucleotide sequences.Further, not all homologies within the Alu-like MN genomic sequences areso close to Alu repeats as to give a hybridization signal understringent hybridization conditions. The percent of homology between MNAlu-like regions and a standard Alu-J sequence are indicated as follows:

Region of Homology within MN Genomic Sequence SEQ. [SEQ ID NO: 5; ID.FIG. 2A-F] NOS. % Homology to Entire Alu-J Sequence  921-1212 59 89.1%2370-2631 60 78.6% 4587-4880 61 90.1% 6463-6738 62 85.4% 7651-7939 6391.0% 9020-9317 64 69.8% % Homology to One Half of Alu-J Sequence8301-8405 65 88.8% 10040-10122 66 73.2%.

MN Proteins and/or Polypeptides

The phrase “MN proteins and/or polypeptides” (MN proteins/polypeptides)is herein defined to mean proteins and/or polypeptides encoded by an MNgene or fragments thereof. An exemplary and preferred MN proteinaccording to this invention has the deduced amino acid sequence shown inFIG. 1. Preferred MN proteins/polypeptides are those proteins and/orpolypeptides that have substantial homology with the MN protein shown inFIG. 1. For example, such substantially homologous MNproteins/polypeptides are those that are reactive with the MN-specificantibodies of t his invention, preferably the Mabs M75, MN12, MN9 andMN7 or their equivalents.

A “polypeptide” or “peptide” is a chain of amino acids covalently boundby peptide linkages and is herein considered to be composed of 50 orless amino acids. A “protein” is herein defined to be a polypeptidecomposed of more than 50 amino acids. The term polypeptide encompassesthe terms peptide and oligopeptide.

MN proteins exhibit several interesting features: cell membranelocalization, cell density dependent expression in HeLa cells,correlation with the tumorigenic phenotype of HeLa×fibroblast somaticcell hybrids, and expression in several human carcinomas among othertissues. MN protein can be found directly in tumor tissue sections butnot in general in counterpart normal tissues (exceptions noted infra asin normal gastric mucosa and gallbladder tissues). MN is also expressedsometimes in morphologically normal appearing areas of tissue specimensexhibiting dysplasia and/or malignancy. Taken together, these featuressuggest a possible involvement of MN in the regulation of cellproliferation, differentiation and/or transformation.

It can be appreciated that a protein or polypeptide produced by aneoplastic cell in vivo could be altered in sequence from that producedby a tumor cell in cell culture or by a transformed cell. Thus, MNproteins and/or polypeptides which have varying amino acid sequencesincluding without limitation, amino acid substitutions, extensions,deletions, truncations and combinations thereof, fall within the scopeof this invention. It can also be appreciated that a protein extantwithin body fluids is subject to degradative processes, such as,proteolytic processes; thus, MN proteins that are significantlytruncated and MN polypeptides may be found in body fluids, such as,sera. The phrase “MN antigen” is used herein to encompass MN proteinsand/or polypeptides.

It will further be appreciated that the amino acid sequence of MNproteins and polypeptides can be modified by genetic techniques. One ormore amino acids can be deleted or substituted. Such amino acid changesmay not cause any measurable change in the biological activity of theprotein or polypeptide and result in proteins or polypeptides which arewithin the scope of this invention, as well as, MN muteins.

The MN proteins and polypeptides of this invention can be prepared in avariety of ways according to this invention, for example, recombinantly,synthetically or otherwise biologically, that is, by cleaving longerproteins and polypeptides enzymatically and/or chemically. A preferredmethod to prepare MN proteins is by a recombinant means. Particularlypreferred methods of recombinantly producing MN proteins are describedbelow for the GST-MN, MN 20-19, MN-Fc and MN-PA proteins.

Recombinant Production of MN Proteins and Polypeptides

A representative method to prepare the MN proteins shown in FIG. 1 orfragments thereof would be to insert the full-length or an appropriatefragment of MN cDNA into an appropriate expression vector as exemplifiedbelow. In Zavada et al., WO 93/18152, supra, production of a fusionprotein GEX-3X-MN (now termed GST-MN) using the partial cDNA clone(described above) in the vector pGEX-3X (Pharmacia) is described.Nonglycosylated GST-MN (the MN fusion protein MN glutathioneS-transferase) was obtained from XL1-Blue cells.

Zavada et al., WO 95/34650 describes the recombinant production of botha glycosylated MN protein expressed from insect cells and anonglycosylated MN protein expressed from E. coli using the expressionplasmid pEt-22b [Novagen Inc.; Madison, Wis. (USA)]. Recombinantbaculovirus express vectors were used to infect insect cells. Theglycosylated MN 20-19 protein was recombinantly produced inbaculovirus-infected sf9 cells [Clontech; Palo Alto, Calif. (USA)]. TheMN 20-19 protein misses the putative signal peptide (aas 1-37) of SEQ IDNO: 6 (FIG. 1), has a methionine (Met) at the N-terminus for expression,and a Leu-Glu-His-His-His-His-His-His [SEQ. ID NO.: 22] added to theC-terminus for purification.

In order to insert the portion of the MN coding sequence for the GST-MNfusion protein into alternate expression systems, a set of primers forPCR was designed. The primers were constructed to provide restrictionsites at each end of the coding sequence, as well as in-frame start andstop codons. The sequences of the primers, indicating restriction enzymecleavage sites and expression landmarks, are shown below.

Primer #20: N-terminus [SEQ. ID. NO. 17]                       

Translation start 5′GTCGCTAGCTCCATGGGTCATATGCAGAGGTTGCCCCGGATGCAG 3′      NheI NcoI    NdeI     

MN cDNA #1 Primer #19: C-terminus [SEQ. ID. NO. 18]            

TransIation stop 5′GAAGATCTCTTACTCGAGCATTCTCCAAGATCCAGCCTCTAGG 3′    BgIII     XhoI 

MN cDNAThe SEQ ID NOS: 17 and 18 primers were used to amplify the MN codingsequence present in the GEX-3X-MN vector using standard PCR techniques.The resulting PCR product (termed MN 20-19) was electrophoresed on a0.5% agarose/1X TBE gel; the 1.3 kb band was excised; and the DNArecovered using the Gene Clean II kit according to the manufacturer'sinstructions [Bio101; LaJolla, Calif. (USA)].

Identification of MN Protein Partner(s)

A search for protein(s) interacting with MN was initiated usingexpression cloning of the corresponding cDNA(s) and a MN-Fc fusionprotein as a probe. The chimerical MN-Fc cDNA was constructed in pSG5Cvector by substitution of MN cDNA sequences encoding both thetransmembrane anchor and the intracellular tail of MN protein with thecDNA encoding Fc fragment of the mouse IgG. The Fc fragment cDNA wasprepared by RT-PCR from the mouse hybridoma producing IgG2a antibody.

The chimerical MN-Fc cDNA was expressed by transient transfection in COScells. COS cells were transfected using lipofection. Recombinant MN-Fcprotein was released to TC medium of the transfected cells (due to thelack of the transmembrane region), purified by affinity chromatographyon a Protein A Sepharose and used for further experiments.

Protein extracts from mock-transfected cells and the cells transfectedwith pSG5C-MN-Fc were analysed by immunoblotting using the M75 MAb,SwαM-Px and ECL Detection® [ECL®—enhanced chemoluminescent system todetect phosphorylated tyrosine residues; Amersham; Arlington, Hts., Ill.(USA)]. The size of MN-Fc protein expressed from the pSG5C vectorcorresponds to its computer predicted molecular weight.

³⁵S-labeled MN-Fc protein was employed in cell surface binding assay. Itwas found to bind to several mammalian cells, e.g., HeLa, Raji, COS,QT35, BL3. Similar results were obtained in cell adhesion assay usingMN-Fc protein dropped on bacterial Petri dishes. These assays revealedthat KATO III human stomach adenocarcinoma cell line is lacking anability to interact with MN-Fc protein. This finding allowed us to useKATO III cells for expression cloning and screening of the cDNA codingfor MN-binding protein.

The cDNA expression library in pBK-CMV vector was prepared from denseHeLa cells and used for transfection of KATO III cells. For the firstround of screening, KATO III cells were transfected by electroporation.After two days of incubation, the ligand-expressing cells were allowedto bind to MN-Fc protein, then to Protein A conjugated with biotin andfinally selected by pulling down with streptavidin-coated magneticbeads. Plasmid DNA was extracted from the selected cells and transformedto E. coli. Individual E. coli colonies were picked and pools of 8-10clones were prepared. Plasmid DNA from the pools was isolated and usedin the second round of screening.

In the second round of screening, KATO III cells were transfected byDEAE dextran method. To identify the pool containing the cDNA forMN-binding protein, an ELISA method based on the binding of MN-Fc to thetransfected cells, and detection using peroxidase labelled Protein Awere used. Pools are selected by ability to bind MN-Fc.

In the third round of screening, plasmid DNAs isolated from individualbacterial colonies of selected pools are transfected to KATO III cells.The transfected cells are subjected to binding with MN-Fc and detectionwith Protein A as before. Such exemplary screening is expected toidentify a clone containing the cDNA which codes for the putative MNprotein partner. That clone would then be sequenced and the expressionproduct confirmed as binding to MN protein by cell adhesion assay.(Far-Western blofting, co-precipitation etc.) Hybridomas producing Mabsto the expression product would then be prepared which would allow theanalysis of the biological characteristics of the protein partner of MN.

Preparation of MN-Specific Antibodies

The term “antibodies” is defined herein to include not only wholeantibodies but also biologically active fragments of antibodies,preferably fragments containing the antigen binding regions. Furtherincluded in the definition of antibodies are bispecific antibodies thatare specific for MN protein and to another tissue-specific antigen.

Zavada et al., WO 93/18152 and WO 95/34650 describe in detail methods toproduce MN-specific antibodies, and detail steps of preparingrepresentative MN-specific antibodies as the M75, MN7, MN9, and MN12monoclonal antibodies. Preferred MN antigen epitopes comprise: aa 62-67(SEQ ID NO: 10); aa 61-66, aa 79-84, aa 85-90 and aa 91-96 (SEQ ID NO:98); aa 62-65, aa 80-83, aa 86-89 and aa 92-95 (SEQ ID NO: 99); aa62-66, aa 80-84, aa 86-90 and aa 92-96 (SEQ ID NO: 100); aa 63-68 (SEQID NO: 101); aa 62-68 (SEQ ID NO: 102); aa 82-87 and aa 88-93 (SEQ IDNO: 103); aa 55-60 (SEQ ID NO: 11); aa 127-147 (SEQ ID NO: 12); aa 36-51(SEQ ID NO: 13); aa 68-91 (SEQ ID NO: 14); aa 279-291 (SEQ ID NO: 15);and aa 435-450 (SEQ ID NO: 16). Example 2 provides further descriptionconcerning preferred MN antigen epitopes.

Bispecific Antibodies. Bispecific antibodies can be produced bychemically coupling two antibodies of the desired specificity.Bispecific MAbs can preferably be developed by somatic hybridization of2 hybridomas. Bispecific MAbs for targeting MN protein and anotherantigen can be produced by fusing a hybridoma that produces MN-specificMAbs with a hybridoma producing MAbs specific to another antigen. Forexample, a cell (a quadroma), formed by fusion of a hybridoma producinga MN-specific MAb and a hybridoma producing an anti-cytotoxic cellantibody, will produce hybrid antibody having specificity of the parentantibodies. [See. e.g., Immunol. Rev. (1979); Cold Spring HarborSymposium Quant. Biol., 41: 793 (1977); van Dijk et al., Int. J. Cancer,43: 344-349 (1989).] Thus, a hybridoma producing a MN-specific MAb canbe fused with a hybridoma producing, for example, an anti-T3 antibody toyield a cell line which produces a MN/T3 bispecific antibody which cantarget cytotoxic T cells to MN-expressing tumor cells.

It may be preferred for therapeutic and/or imaging uses that theantibodies be biologically active antibody fragments, preferablygenetically engineered fragments, more preferably genetically engineeredfragments from the V_(H) and/or V_(L) regions, and still more preferablycomprising the hypervariable regions thereof. However, for sometherapeutic uses bispecific antibodies targeting MN protein andcytotoxic cells would be preferred.

Epitopes

The affinity of a MAb to peptides containing an epitope depends on thecontext, e.g. on whether the peptide is a short sequence (4-6 aa), orwhether such a short peptide is flanked by longer aa sequences on one orboth sides, or whether in testing for an epitope, the peptides are insolution or immobilized on a surface. Therefore, it would be expected byones of skill in the art that the representative epitopes describedherein for the MN-specific MAbs would vary in the context of the use ofthose MAbs.

The term “corresponding to an epitope of an MN protein/polypeptide” willbe understood to include the practical possibility that, in someinstances, amino acid sequence variations of a naturally occurringprotein or polypeptide may be antigenic and confer protective immunityagainst neoplastic disease and/or anti-tumorigenic effects. Possiblesequence variations include, without limitation, amino acidsubstitutions, extensions, deletions, truncations, interpolations andcombinations thereof. Such variations fall within the contemplated scopeof the invention provided the protein or polypeptide containing them isimmunogenic and antibodies elicited by such a polypeptide or proteincross-react with naturally occurring MN proteins and polypeptides to asufficient extent to provide protective immunity and/or anti-tumorigenicactivity when administered as a vaccine.

Epitope for M75 MAb

The M75 epitope is considered to be present in at least two copieswithin the 6× tandem repeat of 6 amino acids [aa 61-96 (SEQ ID NO: 97)]in the proteglycan domain of the MN protein. Exemplary peptidesrepresenting that epitope depending on the context may include thefollowing peptides from that tandem repeat: EEDLPS (SEQ ID NO: 10; aa62-67); GEEDLP (SEQ ID NO: 98; aa 61-66; aa 79-84; aa 85-90; aa 91-96);EEDL (SEQ ID NO: 99; aa 62-65; aa 80-83; aa 86-89; aa 92-95); EEDLP (SEQID NO. 100; aa 62-66; aa 80-84; aa 86-90; aa 92-96); EDLPSE (SEQ ID NO:101; aa 63-68); EEDLPSE (SEQ ID NO: 102; aa 62-68); and DLPGEE (SEQ IDNO: 103; aa 82-87, aa 88-93).

Three synthetic peptides from the deduced aa sequence for the EC domainof the MN protein shown in FIG. 1 were prepared. Those syntheticpeptides are represented by aa 51-72 (SEQ ID NO: 104), aa 61-85 (SEQ IDNO: 105) and aa 75-98 (SEQ ID NO.: 106). Each of those syntheticpeptides contains the motif EEDLP (SEQ ID NO: 100) and were shown to bereactive with the M75 MAb.

Other Epitopes

Mab MN9. Monoclonal antibody MN9 (Mab MN9) reacts to the same epitope asMab M75, as described above. As Mab M75, Mab MN9 recognizes both theGST-MN fusion protein and native MN protein equally well.

Mabs corresponding to Mab MN9 can be prepared reproducibly by screeninga series of mabs prepared against an MN protein/polypeptide, such as,the GST-MN fusion protein, against the peptides representing the epitopefor Mabs M75 and MN9. Alternatively, the Novatope system [Novagen] orcompetition with the deposited Mab M75 could be used to select mabscomparable to Mabs M75 and MN9.

Mab MN12. Monoclonal antibody MN12 (Mab MN12) is produced by the mouselymphocytic hybridoma MN 12.2.2 which was deposited under ATCC HB 11647.Antibodies corresponding to Mab MN12 can also be made, analogously tothe method outlined above for Mab MN9, by screening a series ofantibodies prepared against an MN protein/polypeptide, against thepeptide representing the epitope for Mab MN12. That peptide is aa 55-aa60 of FIG. 1 [SEQ ID NO: 11]. The Novatope system could also be used tofind antibodies specific for said epitope.

Mab MN7. Monoclonal antibody MN7 (Mab MN7) was selected from mabsprepared against nonglycosylated GST-MN as described above. Itrecognizes the epitope represented by the amino acid sequence from aa127 to aa 147 [SEQ ID NO: 12] of the FIG. 1 MN protein. Analogously tomethods described above for Mabs MN9 and MN12, mabs corresponding to MabMN7 can be prepared by selecting mabs prepared against an MNprotein/polypeptide that are reactive with the peptide having SEQ ID NO:12, or by the stated alternative means.

MN-Specific Intrabodies—Targeted Tumor Killing Via IntracellularExpression of MN-Specific Antibodies to Block Transport of MN Protein toCell Surface

The gene encoding antibodies can be manipulated so that theantigen-binding domain can be expressed intracellularly. Such“intrabodies” that are targeted to the lumen of the endoplasmicreticulum provide a simple and effective mechanism for inhibiting thetransport of plasma membrane proteins to the cell surface. [Marasco, W.A., “Review—Intrabodies: turning the humoral immune system outside in orintracellular immunization,” Gene Therapy, 4: 11-15 (1997); Chen et al.,“Intracellular antibodies as a new class of therapeutic molecules forgene therapy,” Hum. Gene Ther., 5(5): 595-601 (1994); Mhashilkar et al.,EMBO J., 14: 1542-1551 (1995); Mhashilkar et al., J. Virol., 71:6486-6494 (1997); Marasco (Ed.), Intrabodies: Basic Research andClinical Gene Therapy Applications, (Springer Life Sciences 1998; ISBN3-540-64151-3) (summarizes preclinical studies from laboratoriesworldwide that have used intrabodies); Zanetti and Capra (Eds.),“Intrabodies: From Antibody Genes to Intracellular Communication,” TheAntibodies: Volume 4, [Harwood Academic Publishers; ISBN 90-5702-559-0(December 1997)]; Jones and Marasco, Advanced Drug Delivery Reviews, 31(1-2): 153-170 (1998); Pumphrey and Marasco, Biodrugs, 9(3): 179-185(1998); Dachs et al., Oncology Res., 9(6-7); 313-325 (1997); Rondon andMarasco, Ann. Rev. Microbiol., 51: 257-283 (1997)]; Marasco, W. A.,Immunotechnology, 1(1): 1-19 (1995); and Richardson and Marasco, Trendsin Biotechnology, 13(8): 306-310 (1995).]

MN-specific intrabodies may prevent the maturation and transport of MNprotein to the cell surface and thereby prevent the MN protein fromfunctioning in an oncogenic process. Antibodies directed to MN's EC, TMor IC domains may be useful in this regard. MN protein is considered tomediate signal transduction by transferring signals from the EC domainto the IC tail and then by associating with other intracellular proteinswithin the cell's interior. MN-specific intrabodies could disrupt thatassociation and perturb that MN function.

Inactivating the function of the MN protein could result in reversion oftumor cells to a non-transformed phenotype. [Marasco et al. (1997),supra.] Antisense expression of MN cDNA in cervical carcinoma cells, asdemonstrated herein, has shown that loss of MN protein has led to growthsuppression of the transfected cells. It is similarly expected thatinhibition of MN protein transport to the cell surface would havesimilar effects. Cloning and intracellular expression of the M75 MAb'svariable region is to be studied to confirm that expectation.

Preferably, the intracellularly produced MN-specific antibodies aresingle-chain antibodies, specifically single-chain variable regionfragments or scFv, in which the heavy- and light-chain variable domainsare synthesized as a single polypeptide and are separated by a flexiblelinker peptide, preferably (Gly₄-Ser)₃ [SEQ ID NO: 116].

MN-specific intracellularly produced antibodies can be usedtherapeutically to treat preneoplastic/neoplastic disease bytransfecting preneoplastic/neoplastic cells that are abnormallyexpressing MN protein with a vector comprising a nucleic acid encodingMN-specific antibody variable region fragments, operatively linked to anexpression control sequence. Preferably said expression control sequencewould comprise the MN gene promoter.

Antibody-Mediated Gene Transfer Using MN-Specific Antibodies or Peptidesfor Targeting MN-Expressing Tumor Cells

An MN-specific antibody or peptide covalently linked to polylysine, apolycation able to compact DNA and neutralize its negative charges,would be expected to deliver efficiently biologically active DNA into anMN-expressing tumor cell. If the packed DNA contains the HSVtk geneunder control of the MN promoter, the system would have doublespecificity for recognition and expression only in MN-expressing tumorcells. The packed DNA could also code for cytokines to induce CTLactivity, or for other biologically active molecules. The M75 MAb (or,for example, as a single chain antibody, or as its variable region) isexemplary of such a MN-specific antibody.

The following examples are for purposes of illustration only and are notmeant to limit the invention in any way.

Example 1 Transient Transformation of Mammalian Cells by MN Protein

This example (1) examines the biological consequences of transfectinghuman or mouse cells with MN-cDNA inserted into expression vectors,mainly from the viewpoint of the involvement of MN protein inoncogenesis; (2) determines if MN protein exerts carbonic anhydraseactivity, and whether such activity is relevant for morphologictransformation of cells; and (3) tests whether MN protein is a celladhesion molecule (CAM).

Synopsis

Methods: MN-cDNA was inserted into 3 expression vectors and was used fortransfecting human or mouse cells. MN protein was detected by Westernblotting, radioimmunoassay or immunoperoxidase staining; in all teststhe MN-specific monoclonal antibody M75 (MAb M75) was used. Carbonicanhydrase activity was determined by the acidification velocity ofcarbonate buffer in CO₂ atmosphere.

Results: (1) Cells (human CGL-1 and mouse NIH3T3 cells) transfected withMN-cDNA showed morphologic transformation, but reverted to normalphenotype after 4-5 weeks. (2) This reversion was not due to the loss,silencing or mutation of the MN insert. (3) MN protein has the enzymeactivity of a carbonic anhydrase, which can be inhibited withacetazolamide; however, the inhibition of the carbonic anhydrase enzymeactivity did not affect transformation. (4) MN protein is an adhesionprotein, involved in cell-to-cell contacts.

Background

This example concerns transformation of mammalian cells by MN-cDNAinserted into expression vectors derived from retroviruses. Such vectorsare suitable for efficient and stable integration into cellular DNA andfor continuous expression of MN protein. Cells transfected with theseconstructs showed morphologic transformation, but after some time, theyreverted to normal phenotype.

Sulfonamides, including acetazolamide, are very potent inhibitors ofknown carbonic anhydrases [Maren and Ellison, Mol. Pharmacol., 3:503-508 (1967)]. Acetazolamide was tested to determine if it inhibitedalso the MN-carbonic anhydrase, and if so, whether inhibition of theenzyme affected cell transformation.

There are reasons to believe that MN protein could be involved in directcell-to-cell interactions: A) previous observations indicated afunctional resemblance of MN protein to surface glycoproteins ofenveloped viruses, which mediate virus adsorption to cell surfacereceptors, and MN participated in the formation of phenotypically mixedvirions of vesicular stomatitis virus. B) Inducibility of MN proteinexpression by growing HeLa cells in densely packed monolayers suggeststhat it may be involved in direct interactions between cells. C)Finally, there is a structural similarity between the MN protein andreceptor tyrosine phosphatase β, which also contains proteoglycan andcarbonic anhydrase domains; those domains mediate direct contactsbetween cells of the developing nervous system [Peles et al., Cell, 82:251-260 (1995)]. Therefore, MN protein was tested to see if it bound tocell surface receptors; the result was clearly positive that it does.

Materials and Methods

Cell Lines

Cells used in this example were: CGL1 and CGL3—respectivelynon-tumorigenic and tumorigenic HeLa×fibroblast hybrids [Stanbridge etal., Somat. Cell Genet., 7: 699-712 (1981)], mouse cell line NIH3T3,HeLa cells and monkey Vero cells. The NIH3T3 cells were seeded at verylow density to obtain colonies started from single cells. The mostnormal appearing colony, designated subclone 2, was picked for use inthe experiments reported in this example.

Expression Vectors

Full-length MN cDNA was acquired from a pBluescript subclone [Pastoreket al., Oncogene, 9: 2877-2888 (1994)]. To remove 5′ and 3′ noncodingsequences, that might reduce subsequent gene expression, a polymerasechain reaction (PCR) was performed. The 5′ primerTAGACAGATCTACGATGGCTCCCCTGTGCCCCAG [SEQ ID NO: 88] encompasses atranslation start site and Bg1II cloning site, and the 3′ primerATTCCTCTAGACAGTTACCGGCTCCCCCTCAGAT [SEQ ID NO: 89] encompasses a stopcodon and XbaI cloning site. Full-length MN-cDNA as a template and PfuDNA Polymerase [Stratagene; LaJolla, Calif. (USA)] were used in thereaction.

The PCR product was sequenced and found to be identical with thetemplate; it carried no mutations. The PCR product harbouring solely theMN coding sequence was inserted into three vectors: 1. pMAMneo[Clontech; Palo Alto, Calif. (USA)] plasmid allowingdexamethasone-inducible expression driven by the MMTV-Long TerminalRepeat (LTR) promoter and containing a neo gene for selection oftransformants in media supplemented with Geneticin (G418) antibiotics.2. Retroviral expression vector pGD [Daley et al., Science, 247: 824-829(1990); kindly provided by Prof. David Baltimore, New York-Cambridge)]containing MLV-LTR promoter and neo gene for G418 antibiotics selection.3. Vaccinia virus expression vector pSC11 [Chakrabarti et al., Mol.Cell. Biol., 5: 3403-3409 (1985)]. Transfection was performed via acalcium-phosphate precipitate according to Sambrook et al. (eds.),Molecular cloning. A laboratory manual, 2nd ed., Cold Spring HarborLaboratory Press (1989).

Vaccinia virus strain Praha clone 13 was used as parental virus[Kutinova et al., Vaccine, 13: 487-493 (1995)]. Vaccinia virusrecombinant was prepared by a standard procedure [Perkus et al.,Virology, 152: 285-297 (1986)]. Recombinant viruses were selected andplaque purified twice in rat thymidine-kinase-less RAT2 cells [Topp, W.C., Virology, 113: 408-411 (1981)] in the presence of5′-bromodeoxyuridine (100 μg/ml). Blue plaques were identified byoverlaying with agar containing5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-Gal) (200 μg/ml).

CA Assay

Carbonic anhydrase activity was measured by a micro-method [Brion etal., Anal. Biochem., 175: 289-297 (1988)]. In principle, velocity of thereaction CO₂+H₂O→H₂CO₃ is measured by the time required foracidification of carbonate buffer, detected with phenol red as a pHindicator. This reaction proceeds even in absence of the enzyme, witht₀=control time (this was set to 60 seconds). Carbonic anhydrase reducesthe time of acidification to t; one unit of the enzyme activity reducesthe time to one half of control time: t/t₀=1/2.

For the experiment, MN protein was immunoprecipitated with Mab M75 fromRIPA buffer (1% Triton X-100, 0.1% deoxycholate, 1 mMphenylmethylsulfonyl-fluoride and 200 trypsin-inhibiting units/ml ofTrasylol in PBS, pH 7.2) extract of Vero cells infected with vaccinia-MNconstruct, after the cells developed cytopathic effect, or with “empty”vaccinia as a control. The MN+antibody complex was subsequently adsorbedto protein A—Staphylococcus aureus cells [Kessler, S. W., J. Immunol.,115: 1617-1624 (1975)] and rinsed 2× with PBS and 2× with 1 mM carbonatebuffer, pH 8.0. The precipitate was resuspended in the same buffer andadded to the reaction mixture. Acetazolamide (Sigma) was tested forinhibition of carbonic anhydrase [Maren and Ellison, supra]. In extractsof infected cells used for immunoprecipitation, the concentration oftotal proteins was determined by the Lowry method [Lowry et al., J.Biol. Chem., 193: 265-275 (1951)] and that of MN protein by acompetition radioimmunoassay as described in Zavada et al., Int. J.Cancer, 54: 268-274 (1993).

Western Blots

Western blotting and development of the blots using ¹²⁵I-labelled M75and autoradiography was performed as before [Pastorekova et al.,Virology, 187: 620-626 (1992); and Zavada (1993), supra].

Adhesion Assay

For the adhesion assay [Hoffman S., “Assays of cell adhesion,” IN:Cell-cell Interactions, (Stevenson et al. eds.) pp. 1-30 (IRL Press atOxford University Press; Oxford, N.Y., Tokyo; 1992)], 25 μl aliquots MNprotein (affinity purified pGEX-3X MN) [Zavada et al. (1993), supra] orof control proteins were spotted on 5 cm-diameter bacteriological Petridishes and allowed to bind for 2 hours at room temperature. This yieldedcircular protein-coated areas of 4-5 mm diameter. MN protein was dilutedto 10 μg/ml in 50 mM carbonate buffer, pH 9.2. Patches of adsorbedcontrol proteins were prepared similarly. Those included collagens typeI and IV, fibronectin, laminin and gelatin (Sigma products), diluted andadsorbed according to the manufacturer's recommendations; FCS and BSAwere also included. After aspiration of the drops, the dishes wererinsed 2× with PBS and saturated for 1 hour with DMEM supplied with 5%FCS. The plates were seeded with 5×10⁵ cells in 5 ml of DMEM+5% FCS andincubated overnight at 37° C. The plates were rinsed with PBS, and theattached cells were fixed with formaldehyde, post-fixed with methanoland Giemsa stained.

Results

39. Transformation and Reversion of CGL1 Cells Transfected with MN-cDNA

Since the expression of MN protein correlated with the tumorigenicity ofHeLa×fibroblast hybrids [Zavada et al. (1993), supra], thenon-tumorigenic hybrid CGL1 cells were first tested. Those cells,transfected with the pMAM.MN construct, after selection with Geneticin,formed colonies with varying degrees of transformation; some of themappeared normal. While normal CGL1 cells are contact inhibited, growingin a parallel orientation, the transformed cells formed very densecolonies, showing the loss of contact inhibition. Such colonies grewmore slowly than the original CGL 1.

After subcloning, the cells isolated from transformed coloniessegregated revertants. The reversion was a gradual, step-wise process;there were colonies with different degrees of reversion. After 2passages, all the cell population became morphologicallyindistinguishable from normal CGL1. This was due to the reversion ofsome cells and to the selective advantage of the revertants, which grewfaster than the transformed cells. Despite repeated attempts, not evenone single stably transformed cell clone was obtained. No transformedcolonies were found in CGL1 cells transfected with an “empty” pMAMcontrol plasmid. Growth of the CGL1+pMAM.MN revertants in media suppliedwith 5 μg/ml of dexamethasone for 7 days enhanced the production of MNprotein, but the morphology of the cells did not return to transformed.

2. Rescue of Transforming MN From the Revertants

The reversion of MN-transformed cells to normal phenotype could have atleast 4 causes: A) loss of the MN insert; B) silencing of the MN insert,e.g., by methylation; C) mutation of the MN insert; D) activation of asuppressor gene, coding for a product which neutralizes transformingactivity of MN protein; E) loss of a MN-binding protein. To decide amongthose alternatives, the following experiment was designed.

MN-cDNA was inserted into pGD, a vector derived from mouse leukemiavirus—MLV. A defective virus was thereby engineered, which contained theMN gene and the selective marker neo instead of genes coding for viralstructural proteins. With this construct, mouse NIH3T3 cells weretransfected. In media supplied with Geneticin, the cells formed colonieswith phenotypes ranging from strongly transformed to apparently normal.All of the transformed colonies and about 50% of the normal coloniesexpressed MN protein. Contrasting with normal NIH3T3 cells, thetransformants were also able to form colonies in soft agar, reflectiveof the loss of anchorage dependence, characteristic of celltransformation. Upon passaging, the cells isolated from transformedcolonies reverted to normal morphology, and at the same time, they lostthe capacity to form colonies in soft agar, while still expressing theMN protein. This permanent presence of MN protein in revertants ruledout alternatives A) and B) supra, that is, loss or silencing of the MNgene as a cause of reversion.

To decide among the other 3 alternatives, the revertants weresuperinfected with live, replication competent MLV. This virus grows inNIH3T3 cells without any morphologic manifestations, and it works as a“helper” for the pGD.MN construct. Virus progeny from MLV-infectedrevertants represents an artificial virus complex [pGD.MN+MLV]. Thisconsists of 2 types of virions: of standard type MLV particles andvirions containing the pGD.MN genome, enveloped in structural proteinsprovided by the “helper” virus. This virus complex was infectious forfresh NIH3T3 cells; it again induced in them morphologic transformationand the capacity to form agar colonies.

Contrasting with NIH3T3 transfected with pGD.MN, all the colonies ofcells infected with [pGD.MN+MLV] complex, which grew in the presence ofGeneticin, were uniformly transformed and contained MN proteins. Thetransformants once more reverted to normal phenotype although they keptproducing infectious [pGD.MN+MLV] complex, which induced transformationin fresh NIH3T3 cells. This cycle of infection-transformation-reversionwas repeated 3 times with the same result. This ruled out alternativeC)—mutation of MN-cDNA as a cause of reversion.

Normal NIH3T3 cells formed a contact inhibited monolayer of flat cells,which did not stain with Mab M75 and immunoperoxidase. Cells infectedwith [pGD.MN+MLV] complex were clearly transformed: they grew in achaotic pattern and showed loss of contact inhibition. Some of the cellsshowed signs of apoptosis. Two passages later, the cell populationtotally reverted to original phenotype as a result of frequent emergenceof revertants and of their selective advantages (faster growth and ahigher efficiency of plating). In fact, the revertants appeared to growto a somewhat lower saturation density than the original NIH3T3 cells,showing a higher degree of contact inhibition.

The control NIH3T3 cells did not contain any MN protein (Western blot);while both transformed cells and revertants contained the same amountand the same proportion of 54 and 58 kDa bands of MN protein. In anon-reducing gel, MN protein was present in the form of oligomers of 153kDa. Consistently, by competition RIA, approximately 40 ng MN/mg totalprotein was found in both of the transformed cells and revertants.

3. Carbonic Anhydrase Activity and its Inhibition

Since the carbonic anhydrase domain represents a considerable part ofthe MN protein (see FIG. 8), tests were performed to determine whetherit is indeed enzymatically active. Vero cells infected with thevaccinia.MN construct, which contained more of the MN protein than othercells used in the present experiments, served as a source of MN protein.The cells were extracted with RIPA buffer, and MN protein wasconcentrated and partially purified by precipitation with MAb M75 andSAC. The immunoprecipitate was tested for CA activity. 78 μl ofprecipitate contained 1 unit of the enzyme. From the extract, theconcentration of total proteins and of MN protein was determined; 1 unitof enzyme corresponded to 145 ng of MN protein or to 0.83 mg of totalprotein. The immunoprecipitate from Vero cells infected with controlvirus had no enzyme activity. Activity of MN carbonic anhydrase wasinhibited by acetazolamide; 1.53×10⁻⁸M concentration of the drug reducedenzyme activity to 50%.

Preliminary tests showed that confluent cultures of HeLa or of NIH3T3cells tolerated 10⁻⁵-10⁻³M concentration of acetazolamide for 3 dayswithout any signs of toxicity and without any effect on cell morphology.In sparse cultures, 10⁻⁵M acetazolamide did not inhibit cell growth, but10⁻⁴M already caused a partial inhibition. Thus, 10⁻⁵M acetazolamide wasadded to NIH3T3 cells freshly transformed with the [pGD.MN+MLV] complex.After 4 days of incubation, the colonies were fixed and stained. Nodifference was seen between cells growing in the presence or absence ofacetazolamide; both were indistinguishable from correctly transformedNIH3T3 cells. Thus, the enzymatic activity of carbonic anhydrase is notrelevant for the transforming activity of MN protein.

4. Cell Adhesion Assay

To determine whether or not MN protein is a cell adhesion molecule(CAM), adhesion assays were performed in plastic bacteriological Petridishes (not treated for use with tissue culture). Cells do not adhere tothe surfaces of such dishes, unless the dishes are coated with a bindingprotein. NIH3T3 cells adhered, spread and grew on patches of adsorbed MNprotein. Only very few cells attached outside the areas coated with MNprotein.

Other variants of the experiment demonstrated that NIH3T3 cells adheredand spread on patches of adsorbed collagen I and IV, fibronectin andlaminin. NIH3T3 cells did not attach to dots of adsorbed gelatin, FCS orBSA.

CGL1, HeLa and Vero cells also adhered to MN protein, but 3 leukemiacell lines showed no adherence. CGL3 cells, strongly expressing MNprotein adhered less efficiently to MN protein dots then did CGL1. Thepresence of 10⁻⁴M acetazolamide in the media did not affect the celladhesion.

To confirm the specificity of adhesion, MN protein was absorbed with SACloaded with MAb M75 (directed to MN) or MAb M67, directed to anunrelated antigen (Pastorekova et al., supra), before it was applied tothe surface of the Petri dishes. Absorption with the SAC-M75 complextotally abrogated the cell binding activity, whereas absorption withSAC-M67 was without any effect.

Additional Cell Adhesion Results

A shortened MN, missing TM and IC segments, is shed into the medium by5ET1 cells (a HeLa×fibroblast hybrid, analogous to CGL3 cells thatexpress MN protein abundantly) or by Vero cells infected with VVcarrying MN-cDNA with deleted TM and IC sequences. The shed MN proteinwas purified from the media, and tested in cell adhesion assays. Thecells adhered, spread and grew only on the patches covered with adsorbedcomplete MN protein, but not on the dots of MN lacking TM and ICregions. Analogous results have been described also for some otheradhesion molecules. A variety of cells (NIH3T3, CGL1, CGL3, HeLa, XC)attached to MN protein dots suggesting that the MN receptor(s) is commonon the surface of vertebrate cells.

Tests were also performed with extracellular matrix proteins or controlproteins dotted on nitrocellulose. The dot-blots were treated with MNprotein solution. Bound MN protein was detected with MAb M75. MN proteinabsorbed to the dots of collagen I and IV, but not to fibronectin,laminin, gelatine or BSA.

Prospects for therapy. There are many new principles of cancer therapyemploying oncoproteins or molecules that interact with them as targets[Mendelsohn and Lippman, “Principles of molecular cell biology ofcancer: growth factors,” In: DeVita et al., eds., Cancer: principles andpractice of oncology, pp. 114-133 4th ed., Philadelphia: Lippinocott(1993); DeVita et al., eds., Biologic therapy of cancer, 2nd ed.,Philadelphia: Lippinocott (1995)]. The MN protein and at least some ofits ligands (or receptors) appear to be particularly suitable for suchpurposes.

Example 2 Identification of MN's Binding Site

MN protein is a tumor-associated cell adhesion molecule (CAM). Toidentify its binding site, a series of overlapping oligopeptides,spanning the N-terminal domain of the MN protein were synthesized. TheN-terminal domain is homologous to that of proteoglycans and contains atandem repeat of six amino acids.

The series of oligopeptides were tested by the cell adhesion assayprocedure essentially as described above in Example 1. The syntheticoligopeptides were immobilized on hydrophobic plastic surfaces to see ifthey would mediate the attachment, spreading and growth of cells. Alsoinvestigated were whether the oligopeptides or antibodies inhibitedattachment of cells (NIH3T3, HeLa and CGL1) to purified MN proteincoated onto such plastic surfaces. The MN protein was affinity purifiedon agarose covalently linked to sulfonamide, as the MN proteinencompasses a CA domain.

Several of the oligopeptides were found to be biologically active: (i)when immobilized onto the plastic, they mediate attachment of cells(NIH3T3, HeLa and to CGL1); (ii) when added to the media, they competefor attachment to cells with the immobilized MN protein; (iii) theseoligopeptides, present in the media do not inhibit attachment of cellsto TC plastic, but they prevent cell-cell adhesion and formation ofintercellular contacts; (iv) treatment of immobilized MN protein and ofactive peptides with MAb M75 abrogates their affinity for the cells; and(v) the binding site of MN was determined to be closely related oridentical to the epitope for MAb M75, at least two copies of which arelocated in the 6-fold tandem repeat of 6 amino acids [aa 61-96 (SEQ IDNO: 97)] in the proteoglycan-like domain of MN protein.

It was concluded that ectopically expressed MN protein most likelyparticipates in oncogenesis by intervention into normal cell-cellcontacts. MN's binding site represents a potential target for whichtherapeutic agents can be designed.

Materials and Methods

Affinity chromatography of MN/CA IX. MN/CA IX was purified by a singlecycle of adsorption—elution on sulfonamide-agarose, as described forother CAs [Falkbring et al., FEBS Letters, 24: 229 (1972)]. We usedcolumns of p-aminoethylbenzenesulfonamide-agarose (Sigma). Columns withadsorbed MN/CA IX were extensively washed with PBS (NaCl 8.0 g/l, KCl0.2 g/l, KH₂PO₄ 0.2 g/l, Na₂HPO₄ 1.15 g/l, pH=7.2) and eluted with 0.1mM acetazolamide (Sigma). All steps of purification were carried out at0-5° C., pH 7.2, at physiological concentration of salts. Complete MN/CAIX+ was extracted with 1% Triton X-100 in PBS from Vero cells infectedwith vaccinia virus containing an insert of complete coding region ofMN/CA IX as described in Zavada et al., Int. J. Oncol., 10: 857 (1997).Before chromatography, the extract was diluted 1:6 with PBS andcentrifuged for 1 h at 1500×g. Truncated MN/CA IX ΔTM ΔIC was producedfrom an analogous construct except that the 3′ downstream primer for PCRwas: 5′ CGT CTA GAA GGA ATT CAG CTA GAC TGG CTC AGC A 3′ [SEQ ID NO:117]. MN/CA IX Δ was shed into the medium, from which it was affinitypurified after centrifugation as above. All steps of purification weremonitored by dot-blots.

Cells and media. The following cell lines were used: HeLa,CGL1=non-tumorigenic hybrid HeLa×fibroblast, CGL3=tumorigenic segregantfrom this hybrid, NIH3T3 cells=mouse fibroblasts. The origin of thecells and growth media are described in Zavada et al., Int. J. Cancer,54: 268 (1993) and Zavada et al., Int. J. Oncol., 10: 857 (1997). Inaddition, we used also HT29, a cell line derived from colorectalcarcinoma (ATCC No. HBT-38).

Cell adhesion assay. The conditions of the assay are basically asdescribed in Example 1. Briefly, 1 μg/ml of purified MN/CA IX in 50 mMmono/bicarbonate buffer, pH 9.2, was adsorbed in 30 μl drops on thebottom of bacteriological 5 cm Petri dishes for 1.5 hr. Then the dropswere removed by aspiration and the dishes were 3× rinsed with PBS andblocked with 50% FCS in culture medium for 30 min. There were twovariants of the test. In the first one, the whole bottom of the Petridish was blocked with 50% FCS, and the dishes were seeded with 5 ml ofcell suspension (10⁵ cells/ml). After overnight incubation, the cultureswere rinsed with PBS, fixed and stained. In the other variant, only thearea of adsorbed MN/CA IX was blocked and on top of MN/CA IX dots wereadded 30 μl drops of cell suspension in growth medium, containing addedoligopeptides (or control without peptides). After incubation, rinsingand fixation, the cultures were stained with 0.5% Trypan blue in 50 mMTris buffer pH 8.5 for 1 h, rinsed with water and dried. Stained areasof attached cells were extracted with 10% acetic acid, the extractstransferred to 96-well plates and absorbance was measured at 630 nm onmicroplate reader.

ELISA. Purified GST-MN [Zavada et al. (1993), supra] at concentration 10ng/ml in carbonate buffer pH 9.2 was adsorbed for 3 h in Maxisorb strips(NUNC). After washing and blocking (1 h) with 0.05% Tween 20 in PBS, 50μl/well of the antibody+antigen mixtures were added. Final dilution ofMAb 75 ascites fluid was 10⁻⁶; concentration of the peptides variedaccording to their affinity for M75 so as to allow determination of 50%end-point. These mixtures were adsorbed for 1.5 h, followed by washingwith Tween-PBS. Bound antibody was detected by antimouse IgG conjugatewith peroxidase (SwAM-Px, SEVAC, Prague), diluted 1:1000. In the colorreaction OPD (o-phenylenediamine dihydrochloride, Sigma) 1 mg/ml in 0.1M citrate buffer pH 5.0 was used. To this H₂O₂ was added to finalconcentration 0.03%. This system is balanced so as to allow assay forantigen competing for M75 as well as for peptides binding to the epitopeof immobilized GST-MN.

Peptides. The peptides used in this study were prepared by the solidphase method [Merrifield et al., IN: Gutte, B. (ed.), Peptides:Synthesis, Strucures and Applications, pp. 93-169 (San Diego; AcademicPress; 1995)] using the Boc/Bzl strategy. The peptide acids wereprepared on PAM-resin and peptide amides on MeBHA resin. Deprotectionand splitting from the resin was done by liquid hydrogen fluoride. Thepeptides were purified by C18 RP HPLC and characterized by amino acidanalysis and FAB MS spectroscopy.

Western blots. MN/CA IX antigens from PAGE gels were transferred to PVDFmembranes (Immobilon P, Millipore) and developed with M75, followed bySwAM-Px (see above) and diaminobenzidine (Sigma) with H₂O₂. Fordot-blots we used nitrocellulose membranes.

Phage display. Ph.D.-7 Phage Display Peptide Library kit was used forscreening as recommended by manufacturer (New England Biolabs). 96-wellplate was coated with peptide SEQ ID NO: 106. Biopanning was carried outby incubating 2×10¹¹ phage with target coated plate for 1 h. Unboundphages were washed away with TBST (50 mM Tris-HCl pH 7.5, 150 mM NaCl,0.1% Tween-20) and specifically bound phages were eluted with M75antibody (2 μg in 100 μl of TBS/well). Eluted phage was amplified andused for additional binding and amplification cycles to enrich the poolin favour of binding sequence. After 5 rounds, individual clones werepicked, amplified and sequenced using T7 sequencing kit (Pharmacia).

Results

Affinity chromatography of MN/CA IX protein. For purification of MN/CAIX protein we decided to use affinity chromatography onsulfonamide-agarose column, described previously for other CAs[Falkbring et al., supra]. The advantages of this method are simplicityand the fact that the whole procedure is carried out undernon-denaturing conditions. Vaccinia virus vector with an insert of thecomplete MN/CA9 cDNA, or with truncated cDNA (lacking transmembrane andintracellular domains) was employed as a source of MN/CA IX protein.

A single cycle of adsorption—elution yielded relatively pure proteins:MN/CA IX+ gave 2 bands of 54 and 58 kDa, MN/CA IXΔ of 54.5 and 56 kDa.These proteins strongly reacted with MAb M75 on Western blots. Inextracts from HeLa, CGL3 and HT29 the blot revealed 2 bands of the samesize as MN/CA IX+ purified from vaccinia virus construct.

Adhesion of cells to MN/CA IX protein. MN/CA IX immobilized onhydrophobic plastic enabled attachment, spreading and growth of cells.Extremely low concentrations of MN/CA IX corresponding to 1 μg/ml ofpurified protein in adsorption buffer were sufficient to cause thiseffect; other cell adhesion molecules are used in 10-50× higherconcentrations. Only complete MN/CA IX protein was active in celladhesion test, truncated MN/CA IX did not support cell adhesion at allor it showed only a low adhesion activity and in some instances it evenacted as a cell “repellent”.

Treatment of the dots of immobilized MN/CA IX with MAb M75 abrogated itscapacity to attach the cells, but the control MAb M16, irrelevant forMN/CA IX had no effect. Blocking of cell attachment by M75 shows thatthe epitope is identical to or overlapping with the binding site ofMN/CA IX for cell receptors.

Identification of the epitope recognized by Mab M75. Preliminary mappingof M75 epitope employing partial sequences of extracellular parts ofMN/CA9 cDNA expressed from bacterial vectors and tested on Western blotslocated it in PG region For exact mapping, our strategy was tosynthesize partially overlapping oligopeptides of 15-25 aa covering thePG domain and test them in competition ELISA with M75. According to theresults, this was followed by a series of 6-12 aa oligopeptides. A majorpart of the PG domain consists of a 6-fold tandem repeat of 6 aa (aa61-96) [SEQ ID NO: 97]; 4 repeats are identical (GEEDLP) [SEQ ID NO: 98]and 2 contain 2 aa exchanged (SEEDSP [SEQ ID NO: 141] and REEDPP [SEQ IDNO: 142]).

Following are the results of competition ELISA with recombinant MN/CA IXand oligopetides synthesized according to partial sequences of the PGregion. MN/CA IX+ and Δ produced in mammalian cells possessed a higherserological activity than any other protein or peptide included in thisexperiment; fusion protein GST-MN synthesized in bacteria was lessactive. The following peptides span the PG region:GGSSGEDDPLGEEDLPSEEDSPC (aa 51-72) [SEQ ID NO: 104];GEEDLPSEEDSPREEDPPGEEDLPGEC (aa 61-85) [SEQ ID NO: 105];EDPPGEEDLPGEEDLPGEEDLPEVC (aa 75-98) [SEQ ID NO: 106]; andEVKPKSEEEGSLKLE (aa 97-111) [SEQ ID NO: 118]. SEQ ID NOS: 104 and 106caused 50% inhibition at 1 ng/ml. Those 2 oligopeptides are mutuallynon-overlapping, thus the epitope is repeated in both of them. SEQ IDNO: 105 was 1000× less active, probably due to a different conformation.SEQ ID NO: 118 was inactive; thus it does not contain the M75 epitope.

The next step for identifying the epitope was to synthesizeoligopeptides containing all circular permutations of the motif GEEDLP[SEQ ID NO: 98] repeated twice. All 6 of the following dodecapeptides[SEQ ID NOS: 119-124] were serologically active (2 more and 4 less so):GEEDLPGEEDLP [SEQ ID NO: 119]; EEDLPGEEDLPG [SEQ ID NO: 120]; EDLPGEEDLP[SEQ ID NO: 121]; DLPGEEDLPGEE [SEQ ID NO: 122]; LPGEEDLPGEED [SEQ IDNO: 123]; and PGEEDLPGEEDL [SEQ ID NO: 124]. The following series of 7aa sequences, flanked by alanine on both ends were tested: APGEEDLPA[SEQ ID NO: 125]; AGEEDLPGA [SEQ ID NO: 126]; AEEDLPGEA [SEQ ID NO:127]; AEDLPGEEA [SEQ ID NO: 128]; ADLPGEEDA [SEQ ID NO. 129]; andALPGEEDLA [SEQ ID NO: 130]. The results showed that the minimumserologically active sequence is the oligopeptide APGEEDLPA [SEQ ID NO:125]. SEQ ID NOS: 127-130 proved negative in competition at 100 μg/ml.Further, none of the following still shorter oligopeptides (6+2aa)competed in ELISA for M75: AGEEDLPA [SEQ ID NO: 131]; AEEDLPGA [SEQ IDNO: 132]; AEDLPGEA [SEQ ID NO: 133]; ADLPGEEA [SEQ ID NO: 134]; ALPGEEDA[SEQ ID NO: 135]; and APGEEDLA [SEQ ID NO: 136].

In the oligopeptides of SEQ ID NOS: 104, 105, 106 and 118, theC-terminal amino acid was present as an acid, whereas in all the otheroligopeptides, the C-terminal amino acid was present as an amide. It isclear that the affinity between these oligopeptides and MAb M75 verystrongly increases with the size of peptide molecule.

Attempts to demonstrate adhesion of cells to immobilized oligopeptides.Our initial plan was to follow the pioneering work of Piersbacher andRuoslahti, PNAS, 81: 5985 (1984). They linked tested oligopeptides toadsorbed bovine serum albumin by cross-linking agent SPDP(N-succinimidyl 3[pyridylhydro]propionate). This is why we added ontothe C-end of oligopeptides SEQ ID NOS: 104-106 cysteine, which wouldenable oriented linking to adsorbed albumin. We demonstrated linking ofthe peptides directly in Petri dishes by immunoperoxidase staining withM75. Unfortunately, CGL1 or CGL3 cells adhered to control albumintreated with SPDP and blocked with ethanolamine (in place ofoligopeptides) as strongly as to BSA dots with linked oligopeptides. Wewere unable to abrogate this non-specific adhesion. Oligopeptides SEQ IDNOS: 104-106 adsorb only very poorly to bacteriological Petri dishes,thereby not allowing the performance of the cell adhesion assay.

Alternatively, we tested inhibition of cell adhesion to MN/CA IX dots byoligopeptides added to the media together with the cell suspension, asdescribed by Piersbacher and Ruoslahti, supra. All peptides SEQ ID NOS:104-106 and 118-136, were tested at concentrations of 100 and 10 μg/ml.None of them inhibited reproducibly the adhesion of CGL1 cells.

Oligopeptides with affinity to M75 epitope which inhibit cell adhesionto MN/CA IX. As an alternative to monoclonal antibodies, we set out toselect oligopeptides exerting affinity to M75 epitope as well as toMN/CA IX receptor binding site from a phage display library of randomheptapeptides—Ph.D.-7. Our aim was to select phages containing thedesired heptapeptides by panning on immobilized peptide SEQ ID NO: 106and subsequent elution with M75. Eluted phage was multiplied inappropriate bacteria and subjected to 4 more cycles of panning andelution. From the selected phage population, 10 plaques were picked,amplified and the heptapeptide-coding region was sequenced. Only 3heptapeptides were represented. Those three heptapeptides, after addingalanine on both sides, are the following nonapeptides: AKKMKRRKA [SEQ IDNO: 137]; AITFNAQYA [SEQ ID NO: 138]; and ASASAPVSA [SEQ ID NO: 139].The last heptapeptide, synthesized again with added terminal alanines asnonapeptide AGQTRSPLA [SEQ ID NO: 140], was identified by panning onGST-MN and eluted with acetazolamide. This last peptide has affinity tothe active site of MN/CA IX carbonic anhydrase. We synthesized thesepeptides of 7+2 aa and tested them in competition ELISA and in celladhesion inhibition. Both tests yielded essentially consistent results:peptide SEQ ID NO: 138 showed the highest activity, peptide SEQ ID NO:137 was less active, peptide SEQ ID NO: 139 was marginally positive onlyin ELISA, and peptide SEQ ID NO: 140 was inactive. In all of those 4nonapeptides, the C-terminal amino acid was present as amide.

Discussion

Purification of transmembrane proteins like MN/CA IX often posestechnical problems because they tend to form aggregates with othermembrane proteins due to their hydrophobic TM segments. To avoid this,we engineered truncated MN/CA IX ΔICΔTM, which is secreted into themedium. Indeed, truncated MN/CA IX was obtained in higher purity thanMN/CA IX+. Unfortunately, this protein was of little use for ourpurposes, since it was inactive in the cell adhesion assay. Such asituation has also been described for other cell adhesion molecules:their shed, shortened form either assumes an inactive conformation, orit adsorbs to hydrophobic plastic “upside down,” while complete proteinsadsorb by hydrophobic TM segments in the “correct” position.

MN/CA IX protein forms oligomers of 150 kDa, linked by disulfidic bonds.It was not known whether these are homo- or hetero-oligomers, but PAGEand Western blot analysis suggest that these are probablyhomo-oligomers, most likely trimers, since on the gel stained withCoomassie Blue no additional bands of intensity comparable to 2 bandsspecific for MN/CA IX appeared. It is also unlikely that there couldexist an additional protein co-migrating with one of the 2 major MN/CAIX bands, since the intensity of their staining on the gel and onWestern blots is well comparable.

There can be no doubt on the specificity of cell attachment to purifiedMN/CA IX+. It is abrogated by specific MAb M75, at a dilution 1:1000 ofascites fluid. This is a correction to our previous report in Zavada etal., Int. J. Oncol., 10: 857 (1997) in which we observed that MN/CA IXproduced by vaccinia virus vector and fusion protein GST-MN support celladhesion, but we did not realize that GST anchor itself contains anotherbinding site, which is not blocked by M75.

MAb M75 reacts excellently with MN/CA IX under any circumstances—withnative antigen on the surface of living cells, with denatured protein onWestern blots and with antigen in paraffin sections of biopsies fixedwith formaldehyde, suggesting that the epitope is small and contiguous.In competition ELISA the smallest sequence reactive with M75 was 7+2 aa,but the affinity between M75 and tested peptides strongly depended ontheir molecular weight. Complete MN/CA IX was 100,000× more active thanthe smallest serologically active peptide in terms of weight/volumeconcentration. In terms of molar concentration this difference would be150,000,000×. Oligopeptides of intermediate size also showedintermediate activities. It remains to be elucidated whether suchdifferences in activity are due to the conformation depending on thesize of the molecule, or to the fact that complete MN/CA IX containsseveral copies of the epitope, but the smallest molecule only one.

Considering the possibility that the epitope is identical with the celladhesion structure in MN/CA IX, we can understand why we failed todetect inhibition of cell adhesion by the oligopeptides. The bindingsite is just not as simple as the prototype peptide, RGD [Winter, J., INCleland and Craik (eds.), Protein Engineering. Principles and Practice,pp. 349-369 (N.Y.; Wiley-Liss; 1996)].

Naturally, one can argue that the size of MN/CA IX is about the same asof immunoglobulin molecule, and that binding of M75 to its epitope maysterically hinder a different sequence of cell attachment site. Thisobjection has been made unlikely by blocking of both M75 epitope and ofcell binding site by nonapeptides 7+2 aa. That result strongly suggeststhat the epitope and the binding site are indeed identical.

MN/CA IX and its PG region in particular appears to be a potentialtarget molecule for therapy for the following reasons: (i) it is exposedon the cell surface; (ii) it is present in high percentage of certainhuman carcinomas; (iii) it is normally expressed in the mucosa ofalimentary tract which is not accessible to circulating antibodies, incontrast with the tumors; (iv) it is not shed (or only minimally) intothe body fluids; (v) the motif GEEDLP [SEQ ID NO: 98] is repeated 18× onthe surface of every MN/CA IX molecule. Oligopeptide display librariesare being employed in the first steps to develop new drugs [Winter, J.,supra]. Selected oligopeptides can serve as lead compounds for thecomputerized design of new molecules, with additional propertiesrequired from a drug [DeCamp et al., IN Cleland and Craik (eds.), supraat pp. 467-505].

Example 3 Identification of Peptides Binding to MN Protein Using PhageDisplay

(a) To identify peptides that are recognized by MN protein, aheptapeptide phage display library [Ph.D.®-7 Peptide 7-mer Library Kit(phage display peptide library kit); New England Biolabs; Beverly, Mass.(USA)] was screened. In screening the library, a selection process,i.e., biopanning [Parmley and Smith, Gene, 73: 308 (1988); Noren, C. J.,NEB Transcript, 8(1): 1 (1996)] was carried out by incubating the phagesencoding the peptides with a plate coated with MN protein, washing awaythe unbound phage, eluting and amplifying the specifically bound phage.

The target MN protein in this process was a glutathione-S-transferase(GST) MN fusion protein (GST-MN). GST-MN is a recombinantly producedfusion protein expressed from pGEX-3X-MN containing the cDNA for the MNprotein without the signal peptide. GST-MN was produced in bacteriaunder modified cultivation conditions (decreased optical density,decreased temperature). Such cultivation prevented premature terminationof translation and resulted in synthesis of the protein molecules whichwere in vast majority of the full length. The GST-MN protein was usedfor coating of the wells and binding the relevant phages. The boundphages were then eluted by acetazolamide, amplified and used for twoadditional rounds of screening.

After sequencing of several independent phage clones obtained after thethird round of screening, the following heptapeptides were obtained:

(1) GETRAPL (SEQ ID NO: 107) (2) GETREPL (SEQ ID NO: 108) (3) GQTRSPL(SEQ ID NO: 109) (4) GQTRSPL (SEQ ID NO: 109) (5) GQTRSPL(SEQ ID NO: 109) (6) GQTRSPL (SEQ ID NO: 109) (7) GQTRSPL(SEQ ID NO: 109)The heptapeptides show very similar or identical sequences indicatingthat the binding is specific. The fact that phages bearing theseheptapeptides were eluted by acetazolamide, an inhibitor of carbonicanhydrase activity, indicates that the peptides bind to the CA domain ofMN protein.

(b) Analogous screening of the heptapeptide phage display library isdone using collagen I, shown to bind MN protein, for elution of phages.Different peptide(s) binding to different part(s) of the MN proteinmolecule are expected to be identified. After identifying suchMN-binding peptides, the corresponding synthetic peptides shall then beanalysed for their biological effects.

Example 4 Accessibility In Vivo of MN Protein Expressed in Tumor Cellsand in Stomach

Lewis rats (384 g) carrying a BP6 subcutaneous tumor (about 1 cm indiameter) expressing rat MN protein were injected intraperitoneally(i.p.) with ¹²⁵I-M75 Mab (2.5×10⁶ cpm). Five days later, 0.5-1 g piecesof the tumor and organs were weighed and their radioactivity wasmeasured by a gamma counter.

Table 2 summarizes the results. The highest radioactivity was present inthe tumor. Relatively high radioactivity was found in the liver andkidney, apparently reflecting the clearance of mouse IgG from the blood.The stomach contained a relatively low level of radioactivity,indicating that the M75 Mab had only limited access to MN proteinexposed in the gastric mucosa.

TABLE 2 Distribution of radioactivity of ¹²⁵I-M75 in rat organs and inthe tumor Organ cpm/g Kidney 2153 2184 Spleen 653 555 Liver 1993 1880Lung 1183 1025 Blood 1449 Heart 568 477 Stomach 1184 1170 Testis 812 779Tail 647 Tumor 3646 4058 3333 8653 3839

Example 5 FACS Analysis of MN Protein Expression in CGL3 Cells—Apoptosis

A FACS investigation was designed to determine the conditions thatinfluence the synthesis of MN protein and to analyse the cell cycledistribution of MN-positive versus MN-negative cells in a CGL3population stimulated to apoptosis. Previous Western blotting analyseshave shown CGL3 cells to express a relatively high amount of MN proteinunder different cultivation conditions. CGL3 cells are considered aconstitutive producer of MN proteins. However, Western blotting does notrecognize small differences in the level of protein. In contrast FACSallows the detection of individual MN-positive cells, a calculation oftheir percentage in the analysed population, an estimation of the levelof MN protein in the cells, and a determination of the cell cycledistribution.

To study the effect of cultivation conditions on MN expression in CGL3cells, the CGL3 cells were plated in different relative densities andserum concentrations. Three days after plating, the cells werecollected, surface labeled by M75 Mab followed by FITC-conjugatedanti-mouse IgG and immediately analysed by FACS.

The analysis showed that in adherent cells, MN expression is dependenton cell density as is HeLa cells. However, low density cultures stillproduced detectable amounts of MN protein. In low density cultures,serum concentration does not seem to play a role. In relatively highdensity cultures, a decreasing serum concentration resulted in slightlydiminished MN expression, probably due to a lower density that the cellswere able to reach during the three days of cultivation.

The effect of the actual cell density is remarkable, and MN expression(detectable in 15-90% of the cells) represents a very sensitivemonitoring factor. In all experiments, there was about a 5% higherpercentage of cycling cells in the MN-positive part of the population,compared to the MN-negative part. That fact prompted the analysis of thecell cycle distribution of MN-positive CGL3 cells under unfavorablegrowth conditions, that is, after induction of apoptosis.

Apoptosis

CGL3 cells were stimulated to apoptotic death by several drugs,including cycloheximide, actimonycin D and dexamethasone. The FACS studyshowed that the onset of apoptosis is delayed in MN-positive cellssuggesting a protective role of MN in this process. It was also observedthat the induction of apoptosis resulted in the down-regulation of MNexpression in a time-dependent manner. That same phenomenon wasdescribed for Bcl-2 anti-apoptotic protein, and there is existingopinion that the down-regulation of certain regulatory genes duringapoptosis sensitizes the cells to undergo apoptotic death. To prove therole of MN in apoptosis, a similar study with cells transfected by MNcDNA is to be performed.

The preliminary results indicate the possible involvement of MN in thesuppression of apoptosis. The recent view that tumors arise both as aconsequence of increased proliferation and decreased cell death appearsto be consistent with the association of the MN protein with tumors invivo.

Examples Concerning MN/CA 9 and Hypoxia

The following materials and methods were used in Examples 6-9 below.

Construction of Reporter Plasmids

To generate plasmids p-506 and p-173, sequences of the MN/CA 9 genebetween −506 and +43 relative to the transcriptional start site wereamplified by PCR from genomic DNA. PCR products were ligated intopGL3-basic, a promoterless and enhancerless luciferase expression vector(Promega). To generate plasmids p-36, MUTI, and MUT2, complementaryoligonucleotides with ends corresponding to the 5′ restriction cleavageoverhangs of Bg/II and MluI were annealed and ligated intoBg/II/MluI-digested pGL3-basic. Oligonucleotides (sense strand) were:p-36 (forward),5′-cgcgCTCCCCCACCCAGCTCTCGTTTCCAATGCA-CGTACAGCCCGTACACACCG-3′; [SEQ IDNO: 152] MUTI (forward),5′-cgcgCTC-CCCCACCCAGCTCTCGTTTCC-AATGCTTTTACAGCCCGTACACACCG-3′; [SEQ IDNO: 153] MUT2 (forward),5′-cgcgCTCCCCCACCCAGCTCTCGTTTCCAATGC-AAGTACAGCCCGTACACACCG-3′ [SEQ IDNO: 154]. Nucleotides introduced for cloning are lowercase; mutationsare underlined. All MN/CA 9 promoter sequences were confirmed by dideoxysequence analysis.

Transient Expression Assays

Cells at ˜70% confluence in 60-mm dishes were transfected with 1 μg of aluciferase reporter construct and 0.4 μg of control plasmid, pCMV-βgal(Promega), using FuGENE 6 (Roche Diagnostic) according to themanufacturer's instructions. Cells were then incubated at 20% O₂ for 8h, followed by 20% or 0.1% O₂ for 16 h.

Luciferase activity was determined in cell lysates using a commercialassay system (Promega) and a TD-20e luminometer (Turner Designs). βgalactivity in cell lysates was measured usingo-nitrophenyl-β-D-galactopyranoside as substrate in a 0.1 M phosphatebuffer (pH 7.0) containing 10 mM KC1, 1 mM MgSO₄, and 30 mMβ-mercaptoethanol. To correct for viable transfection efficienciesbetween experimental conditions, the luciferase: βgal ratio wasdetermined for each sample. For cotransfection assays, cells alsoreceived 0.1-1 μg each of pCDNA3/HIF-1α or pCDNA3/HIF-2α containing theentire human HIF-1α or HIF-2α open reading frame, respectively.Transfections were balanced with various amounts of pCDNA3 (Invitrogen)and pCDNA3/HIF-α such that all cells received the same total quantity ofDNA.

Example 6 Oxygen-Dependent Function of MN/CA 9 Promoter

To investigate the unusually tight regulation of MN/CA 9 mRNA byhypoxia, the oxygen-dependent function of the MN/CA 9 promoter wastested. In the first set of experiments, luciferase reporter genescontaining ˜0.5 kb of MN/CA 9 5′ flanking sequences (−506 to +43) [SEQID NO: 144] and a deletion to nucleotide −173 (−173 to +43) [SEQ ID NO:151] were tested in transiently transfected HeLa cells. Both constructsshowed very low levels of activity in normoxic cells but were inducedstrongly by hypoxia. By contrast, a similar reporter linked to a minimalSV40 promoter showed no induction by hypoxia.

Example 7 Dependence of MN/CA 9 Promoter on HIF-1

To test whether these responses were dependent on HIF-1, furthertransfections were performed using a CHO mutant cell (Ka13) that isfunctionally defective for the HIF-1α subunit and cannot form the HIF-1transcriptional complex. [Wood et al., “Selection and analysis of amutant cell line defective in the hypoxia-inducible factor-1α subunit(HIF-1α),” J. Biol. Chem., 273: 8360-8368 (1998).] In the CHO wild-typeparental subline C4.5, the −173 nucleotide promoter [SEQ ID NO: 151]conferred 17-fold transcriptional induction by hypoxia. In contrast, inthe HIF-1α-deficient Ka13 subline, this hypoxic induction was absent.Cotransfection of human HIF-1α restored hypoxia-inducible activity tothe MN/CA 9 promoter in the Ka13 cells and increased normoxic activityin both C4.5 and Ka13. In C4.5 and Ka13 cells at 0.1% O₂, luciferaseexpression was increased 1.6- and 17-fold, respectively, bycotransfection of human HIF-1α. Thus, hypoxia-inducible activity of theMN/CA 9 promoter is completely dependent on HIF-1 and stronglyinfluenced by the level of HIF-1α. Activity of the MN/CA 9 promoter inKa13 cells could also be restored by cotransfection of HIF-2α, althoughnormoxic activity was higher and fold induction by hypoxic stimulationwas reduced.

Example 8 Response of Putative MN/CA 9 HRE to Hypoxia

Inspection of the MN/CA9 5′ flanking sequences revealed a consensus HREbeginning 3 by 5′ to the transriptional start site, oriented on theantisense strand, reading 5′-TACGTGCA-3′ [SEQ ID NO: 145]. To test theimportance of this site, a MN/CA 9 minimal promoter was constructedcontaining this sequence (−36 to +14) [SEQ ID NO: 146]. This minimalpromoter retained hypoxia-inducible activity in C4.5 cells but had noinducible activity in Ka13 cells. Absolute levels of activity were lowerin comparison to the −173 nucleotide promoter [SEQ ID NO: 151]construct, being reduced ˜8 fold, indicating that although sequences−173 to −36 amplified promoter activity, responsiveness to hypoxia wasconveyed by the minimal sequence containing the MN/CA 9 HRE.

Example 9 Mutational Analysis of MN/CA9 HRE

To confirm the importance of the MN/CA 9 HRE, two mutations were madewithin its core (antisense strand): a 3-bp substitution from CGT→AAA(MUT1), and a single substitution of G→T (MUT2). Both mutationscompletely ablated hypoxia-inducible activity, although basal activitywas preserved or slightly increased for MUT1.

ATCC DEPOSITS

The materials listed below were deposited with the American Type CultureCollection (ATCC) now at 10810 University Blvd., Manassus, Va.20110-2209 (USA). The deposits were made under the provisions of theBudapest Treaty on the International Recognition of DepositedMicroorganisms for the Purposes of Patent Procedure and Regulationsthereunder (Budapest Treaty). Maintenance of a viable culture is assuredfor thirty years from the date of deposit. The hybridomas and plasmidswill be made available by the ATCC under the terms of the BudapestTreaty, and subject to an agreement between the Applicants and the ATCCwhich assures unrestricted availability of the deposited hybridomas andplasmids to the public upon the granting of patent from the instantapplication. Availability of the deposited strain is not to be construedas a license to practice the invention in contravention of the rightsgranted under the authority of any Government in accordance with itspatent laws.

Deposit Date ATCC # Hybridoma VU-M75 Sep. 17, 1992 HB 11128 MN 12.2.2Jun. 9, 1994 HB 11647 Plasmid A4a Jun. 6, 1995 97199 XE1 Jun. 6, 199597200 XE3 Jun. 6, 1995 97198

The description of the foregoing embodiments of the invention have beenpresented for purposes of illustration and description. They are notintended to be exhaustive or to limit the invention to the precise formdisclosed, and obviously many modifications and variations are possiblein light of the above teachings. The embodiments were chosen anddescribed in order to explain the principles of the invention and itspractical application to enable thereby others skilled in the art toutilize the invention in various embodiments and with variousmodifications as are suited to the particular use contemplated.

All references cited herein are hereby incorporated by reference.

1. A method for determining the percentage of the cells in a vertebratetumor that are hypoxic, comprising: a) administering to said vertebratetumor an expression vector comprising a nucleic acid encoding a reportergene operatively linked to a hypoxia-inducible MN/CA9 promoter fragment;and b) detecting and quantifying reporter gene expression productexpressed by said vector in cells of said tumor or in a tissue sampletaken from said tumor; and c) determining the percentage of hypoxiccells in said tumor based on the percentage of cells expressing saidreporter gene expression product; wherein said hypoxia-inducible MN/CA9promoter fragment comprises a nucleotide sequence selected from thegroup consisting of: (1) SEQ ID NO: 146 and its complement; and (2)nucleotide sequences that are 90% homologous to the nucleotide sequencesof (1); and wherein said hypoxia-inducible MN/CA9 promoter fragment hashypoxia-inducible activity.
 2. A method for determining the relativedegree of hypoxia in a tissue in a vertebrate, comprising: a)administering to said vertebrate tissue an expression vector comprisinga nucleic acid encoding a reporter gene operatively linked to ahypoxia-inducible MN/CA9 promoter fragment; b) detecting MN/CA9 reportergene expression product in said tissue; and c) quantifying said reportergene expression product, wherein the relative level of reporter geneexpression product indicates the relative degree of hypoxia; whereinsaid hypoxia-inducible MN/CA9 promoter fragment comprises a nucleotidesequence selected from the group consisting of: (1) SEQ ID NO: 146 andits complement; and (2) nucleotide sequences that are 90% homologous tothe nucleotide sequences of (1); and wherein said hypoxia-inducibleMN/CA9 promoter fragment has hypoxia-inducible activity.
 3. The methodof claim 2 wherein said hypoxia is chronic hypoxia.
 4. The method ofclaim 2 wherein a higher degree of hypoxia in said tissue is consideredindicative of a poorer prognosis of a preneoplastic/neoplastic disease.5. The method of claim 4, wherein a poorer prognosis is measured interms of shortened survival or increased risk of recurrence of saidpreneoplastic/neoplastic disease.
 6. The method of claim 5 wherein saidmethod is used as an aid in making a therapeutic decision in thetreatment of said preneoplastic/neoplastic disease afflicting saidvertebrate.
 7. The method of claim 6 wherein said method comprisespredicting patient response to a specific therapy or specific therapies.8. The method of claim 6 wherein said therapeutic decision comprises adecision to treat said disease with a hypoxia-modifying therapy ifhypoxia is present in high levels.
 9. The method of claim 6 wherein saidtherapeutic decision comprises a decision to treat said disease withbio-reductive drugs if hypoxia is present in high levels.
 10. The methodof claim 6 wherein said therapeutic decision comprises a decision totreat said disease with radiation and/or chemotherapy if hypoxia isabsent or present in low levels.
 11. The method of claim 6, wherein saidtherapeutic decision comprises a decision to treat said disease with acombination of radiotherapy and hypoxia-directed therapy if hypoxia ispresent in high levels.
 12. The method of claim 6 wherein saidtherapeutic decision comprises a decision to treat said disease moreaggressively if hypoxia is present in high levels in said tissue, orless aggressively if hypoxia is absent or present in low levels in saidtissue.
 13. The method of claim 4, wherein said disease is selected fromthe group consisting of preneoplastic/neoplastic diseases related tohead and neck cancers, head and neck squamous cell carcinoma, uterinecarcinoma, uterine cervical carcinoma, renal cancer, breast cancer, lungcancer, pancreatic cancer, soft tissue sarcoma, bladder cancer,colorectal cancer, esophageal cancer, prostate cancer, ovarian cancer,endometrial cancer, squamous cell and adenosquamous carcinomas,mesodermal tumors, neuroblastomas, retinoblastomas, sarcomas,osteosarcomas, Ewing's sarcoma, melanomas, vaginal cancer, vulvalcancer, gastrointestinal cancer, urinary tract cancer, kidney cancer,non-small cell lung cancer, skin cancer, liver cancer, cervical squamouscell carcinoma, cervical adenosquamous carcinoma, cervicaladenocarcinoma, cervical metaplasia, and condyloma.